Oleosins, which are structural proteins on the surface of intracellular oil bodies, have been found in the sporophytic seeds of angiosperms. Here, we report an oleosin from the female gametophyte of gymnosperm Pinus ponderosa Laws. seed and another oleosin from the male gametophyte of Brassica napus L. With the pine seed gametophyte, we identified two putative oleosins of 15 and 10 kDa, which are similar to the oleosins in angiosperm seeds in terms of their presence in the oil bodies in massive quantity. The complete sequence of the cDNA encoding the gametophytic 15-kDa oleosin was obtained, and it has a predicted amino-acid sequence similar to those of oleosins in angiosperm sporophytic seeds. A Brassica napus pollen cDNA sequence, which was reported earlier, would encode an amino-acid sequence somewhat similar to those of seed oleosins. We tested if the dissimilarity signifies a substantially different oleosin in the Brassica male gametophyte or an analytic error. By direct sequencing of a polymerase chain reaction (PCR)-amplified fragment of genomic DNA, we obtained evidence showing that this reported dissimilarity is likely to have arisen from a sequencing error. Our predicted sequence of the Brassica pollen oleosin has all the structural characteristics of seed oleosins. A phylogenic tree of 20 oleosins, including those from sporophytic and gametophytic tissues of angiosperm and gymnosperm, was constructed based on their amino-acid sequences. We discuss the evolution of oleosins, and conclude that oleosins are ancient proteins with multiple lineages whose root cannot be determined at this time.