Retinol or retinoic acid is required for insulin release. Retinoids increase transglutaminase activity, and transglutaminase has been implicated in islet insulin release. To examine whether transglutaminase could mediate effects of retinoids on insulin secretion, we measured (i) transglutaminase activity in islets from rats deficient in vitamin A or repleted with retinol or retinoic acid, (ii) transglutaminase activity in RINm5F and INS-1 insulin-secreting cells cultured in retinol or retinoic acid, (iii) mRNA for transglutaminase in RINm5F and INS-1 cells, and (iv) insulin secretion from INS-1 cells in response to retinoic acid. Islets from rats repleted with retinol or retinoic acid showed more than twice the transglutaminase activity of islets from vitamin A-deficient rats. Retinoic acid increased RINm5F cells and INS-1 cell transglutaminase activity. Retinol did not increase transglutaminase activity. Transglutaminase mRNA was detected in INS-1 cells but not in RINm5F cells. Retinoic acid increased insulin secretion from INS-1 cells as observed previously in RINm5F cells. In conclusion, retinoic acid increases transglutaminase activity in both rat islets and two insulin-secreting cell lines. Retinoic acid stimulates insulin secretion from INS-1 cells. Transglutaminase is a candidate for mediating retinoid-induced changes in insulin secretion.