The biologic activities of retinoic acid and 3,4-didehydroretinoic acid, two endogenous vitamin A derivatives in various tissues, were compared to their affinities for the nuclear retinoic acid receptors and their ability to induce transcriptional activation. Both retinoids were equipotent inducers of differentiation of F9 teratocarcinoma cells. In a morphologic assay, using reconstructed skin, retinoic acid and 3,4-didehydroretinoic acid inhibited keratinization at a concentration of 100 nM. In cultured keratinocytes, a 50% inhibition of the production of the keratinocyte transglutaminase enzyme was achieved with about 20 nM for both retinoids. The in vitro binding to the nuclear retinoic acid receptors alpha, beta, and gamma showed that retinoic acid and 3,4-didehydroretinoic acid had almost equal affinities for the receptors with Kds ranging from 3 to 47 nM. The transcriptional activation resulting from the addition of the two retinoids to cells co-transfected with alpha, beta, or gamma retinoic acid receptor expression vectors and a retinoic acid responsive element linked to the chloramphenicol acetyltransferase reporter gene was similar. Finally, it was demonstrated that retinoic acid did not metabolize to 3,4-didehydroretinoic acid, and a slow conversion of 3,4-didehydroretinoic acid into retinoic acid was not sufficient to explain the biologic effects produced by the former compound. In conclusion, the present study demonstrates that retinoic acid and 3,4-didehydroretinoic acid have the same activity in several different test systems, but their metabolism differs depending on the cell type used.