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We recently identified a broadly expressed transporter, PGT, that transports primarily prostaglandins E2 and F2 alpha (PGE2 and PGF2 alpha). In the current study, we examined the structural determinants of potential PGT substrates in detail. Rat PGT was transiently expressed in HeLa cells, the timed uptake of tracer PGE2 was determined in the presence of various concentrations of unlabeled prostanoids; and the resulting inhibitory constants (Ki) were determined by curve-fitting. PGE2 and PGF2 alpha, both known to be transported, had similar affinities for PGT (Ki = 49-50 nM). The strongest interaction (Ki = 13-19 nM) was obtained with prostanoids lacking the 9- or 11-position oxygen groups. A relatively high affinity was also obtained for the bicycloendoperoxides U44069, PGH2, and U46619 (Ki = 29-39 nM). However, a radioactive representative from this group, U46619, was not transported. Structural modifications that produced a moderately reduced affinity relative to that of PGE2 (Ki = 56-286 nM) included reduction in C5 = C6, the addition of a benzene group at position C18, and isomerization at the C8 position. In complementary studies, tracer isoprostane B-iso-PGF2 alpha was found to be transported at approximately 13% the rate of tracer PGE2. Substantially weaker interaction (Ki = > 700 nM) was seen when the 1-position COO- anionic group was neutralized or when the 15(S)-OH group was changed to 15(R)-OH or to 15-keto. These results with the cloned rat PGT are very similar to those previously reported in the in vitro perfused rat lung and indicate that PGT probably represents the predominant route by which certain prostanoids, including F2 isoprostanes, are transported across plasma membranes.