Sympathetic innervation is known to increase heart size in the immature animal, yet the mechanism for this growth remains to be established. This comparative study stereologically quantified the volume of cultured neonatal ventricular myocytes with and without in vitro sympathetic innervation to isolate the mechanisms regulating cardiac growth. Since ventricular myocyte size at birth differs between the spontaneously hypertensive rat (SHR) and the normotensive Wistar-Kyoto (WKY), we questioned whether SHR myocytes respond differently than WKY myocytes to innervation. Four groups of ventricular myocytes from each strain were compared: myocytes grown alone, myocytes innervated by cultured sympathetic neurons, innervated myocytes exposed to adrenoceptor blockade, and non-innervated myocytes in co-culture dishes. Volumes for the myocyte, nucleus, cytoplasm, mitochondria, sarcomeres and other cellular organelles were assessed within each population and between populations. Relative volumes were determined for the mitochondria, sarcomeres, and other cellular components within the cytoplasm. Innervated WKY myocytes were 38% larger than control myocytes (P < 0.0004). This growth was not blocked by adrenoceptor blockade (P = 0.89 vs. innervated) and was present in the non-innervated myocytes distant from the neurons in the co-cultures (P = 0.39 vs. innervated). SHR myocytes were 36% larger than WKY myocytes (P < 0.009) but did not increase with innervation (P = 0.48). SHR myocyte size was also unaffected by adrenoceptor blockade (P = 0.39) or presence of the neurons in the culture dish (P = 0.53). Neonatal WKY ventricular myocyte growth can be provoked in vitro by sympathetic innervation via regulatory mechanisms independent of neuroeffector transmission or anatomic contact, whereas volume of neonatal SHR myocytes is unaltered by sympathetic coculture. These findings are significant for understanding normal as well as aberrant cardiomyocyte growth.