Pulse labeling with [35S]-methionine, one-dimensional SDS-polyacrylamide gel electrophoresis and fluorography were used to study the pattern of protein synthesis in Neurospora crassa mycelia undergoing sexual development. 2. Contact of sexually-competent mycelium with cells of the opposite mating type elicited a rapid and transient increase in the synthesis of two predominant proteins of 58 kDa and 40 kDa localized in the cytosol fraction. 3. Marked changes in the pattern of protein synthesis were also observed in the 12,000 g particulate fraction, predominantly mitochondrial, where the synthesis of a 34 kDa polypeptide was most prominent among others. 4. Poly(A)+ RNA extracted from mycelia 2 h after sexual stimulation supported the in vitro synthesis of the 58 kDa and 40 kDa major polypeptides synthesized in vivo. 5. No differences were observed in the pattern of protein synthesis of treated cultures and controls 24 h after the sexual stimulus.