An enriched plasma membrane fraction was isolated from caput, corpus, and cauda rat spermatozoa and analyzed for lipid and protein content, thermal phase transition temperature using electron paramagnetic resonance spectroscopy (EPR), and enzymatic assays of calcium‐dependent AT‐Pase activity. Based on sperm concentration, total membrane phospholipid, cholesterol, and protein content declined as sperm passed through the epididymis. A more refined analysis of the bulk plasma membrane phospholipid revealed that approximately 56% of the phospholipid consisted of choline (PC) and ethanolamine (PE) phosphoglycerides; the remainder consisted of sphingomyelin (SM), phosphatidylserine (PS), and diphosphatidylglycerol (DPG). The mole percent of PE increased in sperm proceeding from the caput to the corpus epididymis and then declined from the corpus to the cauda epididymis. The phospholipid‐bound fatty acids consisted primarily of palmitate (C16:0) and stearate (C18:0), with a significant increase in the mole percent of the docosapentenoyl acyl group (C22:5) in cauda sperm. Arrhenius' plots of the EPR peak height signals using the lipid soluble spin label, 5‐doxyldecane, and the calcium‐dependent ATPase activity as a function of temperature demonstrated a change in the apparent fluidity of the membrane and energy of activation of the calcium‐dependent ATPase associated with the three sperm membrane preparations. These data suggest that the apparent fluidity and biochemical composition of the sperm membrane change during epididymal maturation.