Adult rat testis homogenates were fractionated by differential centrifugation followed by two discontinuous gradient centrifugation steps under identical conditions except for the absence of digitonin in the first gradient and the presence of 0.03% digitonin in the second gradient. The first gradient centrifugation yielded a membrane fraction enriched 28.8-fold in 5'-nucleotidase, 21.5-fold in UDP-Gal:GlcNAc galactosyltransferase and 18.6-fold in UDP-GlcNAc:alpha-D-mannoside N-acetylglucosaminyltransferase. Repeat centrifugation of this membrane fraction in the denser level of the gradient; this material was enriched 32.1-fold in 5'-nucleotidase but only 1.9-fold in galactosyltransferase and 8.4-fold in N-acetylglucosaminyltransferase. The plasma membrane fraction was shown to be free of glucose-6-phosphatase, succinate dehydrogenase, beta-N-acetylglucosaminidase, DNA, and RNA. The fraction therefore appears to be enriched in plasma membrane but relatively free of Golgi membrane contamination, as indicated by the relatively low levels of glycosyltransferases, and of contamination by other organelles. The testicular cells which contribute plasma membrane to this fraction have not yet been definitively identified; the contribution by Sertoli cells is particularly difficult to assess since these cells have been reported to be enriched in 5'-nucleotidase. However, sulfogalactosylalkylacylglycerol (SGG), a lipid previously shown to be present primarily in primary spermatocytes, spermatids, and spermatozoa, was enriched 33.1-fold in the plasma membrane fraction; this finding as well as experiments with [35S]sulfate-labeled sulfogalactosylalkylacylglycerol at various times after injection of radioactive label have indicated that both spermatocytes and spermatids were contributing SGG-rich membrane material to our plasma membrane preparation. This membrane material is most probably derived from the plasma membranes of the spermatocytes and spermatids.