Immunoblot analysis of a rat testis cytosol fraction revealed two proteins which reacted with a polyclonal rabbit antibody to bovine phosphatidylinositol transfer protein. These two proteins were separated by anion exchange and molecular sieve column chromatographic procedures and shown to catalyze the transfer of phosphatidylinositol and phosphatidylcholine between populations of small unilamellar vesicles. One protein was identified as the phosphatidylinositol transfer protein detectable in 16 other rat tissues and many eukaryotic species; the other phosphatidylinositol transfer protein was unique to testis. The molecular masses of the proteins, determined under denaturing electrophoretic conditions, were 35 and 41 kDa, respectively. When testis was examined in animals from birth to six weeks of age, the 35-kDa protein was present throughout, while the 41-kDa protein first appeared during week 4 and increased to adult levels by week 6; a small yet significant increase in tissue phosphatidylinositol transfer activity accompanied this expression of the testis-specific protein. Selective destruction of Leydig cells by ethylene dimethanesulfonate did not cause any detectable loss of the 41-kDa phosphatidylinositol transfer protein. The structural and catalytic relationships between the two testicular phosphatidylinositol transfer protein species remain to be elucidated.