We have been investigating the hypothesis that prostaglandins including prostaglandin E2 (PGE2) produced during the critical condensation phase of limb chondrogenesis are involved in the regulation of cartilage differentiation by acting as local modulators of cyclic AMP (cAMP) accumulation. The purpose of the present study was to determine directly whether PGE2 and other prostanoids which had previously been shown to stimulate in vitro chondrogenic differentiation do indeed elevate the cAMP content of limb mesenchymal cells, and to determine whether the ability of various prostanoids to increase cAMP production by these cells directly reflects the potencies of these same molecules in stimulating chondrogenesis. We have found that PGE2 does indeed elicit a striking elevation in the cAMP content of subridge mesenchymal cells, indicating that the cells possess adenylate cyclase-coupled receptors for this molecule. The effect of PGE2 on cAMP accumulation is potentiated by a phosphodiesterase inhibitor, thus paralleling the potentiating effect phosphodiesterase inhibitors have on PGE2-stimulated in vitro chondrogenesis. The effect of PGE2 on cAMP content is dose-dependent with a 3-fold increase seen at 10(-8)M, which is the lowest concentration at which PGE2 effectively stimulates chondrogenesis. PGE1, which is just as effective as PGE2 in stimulating chondrogenesis, is just as effective as PGE2 in stimulating cAMP accumulation. PGA1, which is a much less effective stimulator of chondrogenesis than PGE2 or PGE1, is less than half as potent as these molecules in elevating cAMP levels. PGF1 alpha, 6-keto PGF1 alpha, and thromboxane B2, which have little or no effect on chondrogenesis, have little or no effect on cAMP content.(ABSTRACT TRUNCATED AT 250 WORDS)