At the in vitro threshold serum concentration of 500 ng/ml, isotretinoin induces defects of visceral arch development in 9.5-day rat embryos grown in culture for 48 h. Experiments were performed to determine the minimum period of exposure necessary to induce these arch defects and whether an increase in concentration of isotretinoin could compensate for reduced exposure time. The results showed that a minimum 6-h exposure to 500 ng/ml immediately prior to cranial neural crest migration was necessary to induce severe defects of the second visceral arch in a majority of embryos. Maximal increase in isotretinoin concentration to 16,000 ng/ml did not compensate for shorter exposure periods. These results suggest that to cause malformations of the visceral arches, the embryo must be exposed to isotretinoin for a minimum period of time regardless of the concentration of isotretinoin above the threshold.