Receptors (Rs) for prostaglandin E2 (PGE2) of the EP2 subtype are expressed at high levels in rodent and human thymuses, with preferential localization on immature thymocytes. Human cultured lymphoblasts of the Tsup-1 line express CD4 and CD8 but only a low level of CD3, typical of immature thymocytes, and bear EP2-type Rs for PGE2 that were identified by binding of [3H]PGE2 and Ab to recombinant EP2Rs, and by cAMP responses to PGE2. PGE2 protected Tsup-1 cells from apoptosis initiated by diverse stimuli, including mitogenic lectins and anti-Fas or anti-CD3 plus anti-CD28 Abs, but not ionomycin, as assessed by suppression of both fragmentation of [3H]thymidine-labeled DNA and appearance of free 3'-OH ends of cleaved DNA. An EP2R-selective synthetic agonist also significantly suppressed lectin-induced apoptosis of Tsup-1 cells. Phosphodiesterase inhibition synergistically enhanced PGE2-induced increases in cAMP and decreases in apoptosis in parallel, which suggests that the EP2R-specific protective effect of PGE2 is mediated predominantly by cAMP suppression of apoptosis. Dibutyrylcyclic AMP alone protected Tsup-1 cells against lectin-induced apoptosis, but the maximal effect was less than that for PGE2. The thromboxane A2 mimetic U46619 initiated apoptosis of Tsup-1 cells that was suppressed significantly by PGE2. Immune negative selection of immature thymocytes thus may be regulated by opposing effects of endogenous eicosanoids that include destruction by thromboxane A2 and protection by PGE2.