The biochemical composition of ejaculated spermatozoal plasma membranes from normal-control (n = 4) and heat-stressed (n = 4) paired, sibling boars was assessed over a 9-week collection period. A total of 36 ejaculates were collected from the normal-control boars. Phospholipids (PL) accounted for 83.5% of the membrane lipid material, with sterols making up the remaining 16.5%. Phosphatidylcholine was the most abundant PL (56.6%), followed by sphingomyelin (16.6%), phosphatidylglycerides (11.0%), lysophosphatidylcholine (9.0%), phosphatidylethanolamine (5.8%), and phosphatidylinositol (1.0%). Cholesterol was the major sterol found in the sperm's plasma membrane (72.1%), with desmosterol being the only other consistently detectable sterol. No difference was found in membrane sterol composition over the 9-week collection period between control boars. A slight fluctuation in membrane PL (P = 0.047) was observed; this was found to be primarily an effect of boar (P = 0.01), than an effect of week (P = 0.26). Phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine fluctuated more (P < 0.05) than did the remaining PL. Membrane protein concentration was different within and between control boars (P < 0.05) throughout the collection period; 19 individual polypeptides were consistently identified ranging from 14 to 111 kDa;Heat-stress treatment for 120 hours (8 hours at 35-37°C, 20-30% relative humidity, followed by 16 hours at 30-32°C, 20-30% relative humidity for 5 consecutive days) had a significant (P < 0.05) effect on membrane lipid composition. Out of a total of 33 ejaculates, membrane sterol composition was the most dramatically reduced, starting at day 7 post-treatment through day 56. Phosphatidylcholine (P < 0.05) and lysophosphatidylcholine (P < 0.01) were significantly reduced, reaching their lowest membrane concentrations 49-56 days post-treatment. No difference in protein concentration was observed between control and treated groups; additionally, the same 19 major polypeptides were identified in the sperm plasma membranes from the treated group;Spermatogenic cell types most sensitive to heat-stress included primary/secondary spermatocytes, round/elongating spermatids, and possibly Type B spermatogonia. Plasma membrane alterations were still present in treated boar spermatozoa even after regaining normal spermiogram parameters (i.e. sperm motility, morphology, and concentration).