The biological activity of various natural retinoids and the time "window" when vitamin A activity is required for normal cardiovascular development were examined in vitamin A-deprived Japanese quail embryos. The administration of 1 microgram of retinol at the beginning of incubation resulted in normal cardiovascular development in 97% of embryos; retinoic acid was toxic at this dose level. Treatment of embryos with 0.1 microgram of all-trans-retinol or 13-cis-retinoic acid at the beginning of incubation resulted in normal cardiovascular development in 47 and 12% of embryos, respectively; administration of these retinoids at other time points attenuated the percentage of embryos with normal cardiovascular development. Didehydroretinol, 0.1 microgram, and 9-cis-retinoic acid, 0.1 microgram, were inactive at all time points examined; 9-cis-retinoic acid did not enhance the biological activity of all-trans-retinoic acid. All-trans-retinoic acid, 0.1 microgram, administered during 22-28 hr of incubation induced normal cardiovascular development in 20-34% of embryos; biological activity was optimal when it was administered at 24 hr. All retinoids tested were inactive in establishing normal cardiovascular development when administered at 36 hr of incubation or later. The studies suggest that all-trans-retinoic acid is the biologically active form of vitamin A required for normal cardiovascular development in the avian embryo. There is a critical time point within the first 22-28 hr of quail embryogenesis when all-trans-retinoic acid initiates events that lead to normal cardiovascular development.