Regional fascicle strains were recorded in vivo from the pectoralis of carneau pigeons using sonomicrometry during level slow flight, together with regional electromyography (EMG) and deltopectoral crest (DPC) strain measurements of whole muscle force. Fascicle strain measurements were obtained at four sites within the pectoralis: the anterior (Ant), middle (Mid) and posterior (Post) sternobrachium (SB), and the smaller thoracobrachium (TB). Strains were also recorded along the intramuscular aponeurosis of the pectoralis to assess its 'in-series' compliance with respect to strains of Post SB and TB fascicles. In-series segment strains were also obtained along Ant SB and Mid SB fascicles, which insert directly on the DPC without attaching to the intramuscular aponeurosis. In-series segment strains differed from 2% to 17.2%, averaging differences of 6.1% at the Ant SB site and 1.4% at the Mid SB site. Temporal patterns of in-series fascicle segment strain were similar at both sites. Regional fascicle strains also exhibited similar temporal patterns of lengthening and shortening and were most uniform in magnitude at the Ant SB, Mid SB and TB sites (total strain: 33.7%, 35.9% and 33.2% respectively), but were smaller at the Post SB site (24.4%). Strains measured along the aponeurosis tracked the patterns of contractile fascicle strain but were significantly lower in magnitude (19.1%). Fascicle lengthening strains (+25.4%) greatly exceeded net shortening strains (-6.5%) at all sites. Much of the variation in regional fascicle strain patterns resulted from variation of in vivo recording sites among individual animals, despite attempts to define consistent regions for obtaining in vivo recordings. No significant variation in EMG activation onset was found, but deactivation of the Ant SB occurred before the other muscle sites. Even so, the range of variation was small, with all muscle regions being activated midway through lengthening (upstroke) and turned off midway through shortening (downstroke). While subtle differences in the timing and rate of fascicle strain may relate to differing functional roles of the pectoralis, regional patterns of fascicle strain and activation suggest a generally uniform role for the muscle as a whole throughout the wingbeat cycle. Shorter fascicles located in more posterior regions of the muscle underwent generally similar strains as longer fascicles located in more anterior SB regions. The resulting differences in fiber length were accommodated by strain in the intramuscular aponeurosis and rotation of the pectoralis insertion with respect to the origin. As a result, longer Ant and Mid SB fascicles were estimated to contribute substantially more work per unit mass than shorter Post SB and TB fascicles. When the mass fractions of these regions are accounted for, our regional fascicle strain measurements show that the anterior regions of the pectoralis likely contribute 76%, and the posterior regions 24%, of the muscle's total work output. When adjusted for mass fraction and regional fascicle strain, pectoralis work averaged 24.7+/-5.1 J kg(-1) (206.6+/-43.5 W kg(-1)) during level slow (approximately 4-5 m s(-1)) flight.