Using intact rat islets, hamster In-R1-G9 cells, and mouse αTC-1 clone 9 transgenic tumoral glucagon-secreting cells, we determined the effects of retinol (ROH) and retinoic acid (RA) on glucagon secretion. Since vitamin A effects may be mediated through nuclear RA receptors (RARs) and cytoplasmic ROH- and RA-binding proteins (CRBP and CRABP), cells were also assayed for RARs, CRBP, and CRABP mRNA by Northern blot analyses. Islets and cells were cultured in 2.8 mmol/L glucose and vitamin A-deficient (A-def) medium or in different concentrations of ROH and RA. Using intact islets, RA 10 and 100 nmol/L inhibited glucagon secretion to approximately 60% of control levels. Using In-R1-G9 cells, ROH 0.175 to 5.0 μmol/L inhibited glucagon secretion to 60% to 83% of control levels, and RA 100 and 1,000 nmol/L inhibited glucagon secretion from 72% to 43% of control levels, respectively. Using uTC-1 cells, ROH 1.75 p mol/L inhibited glucagon secretion to 80% of control levels, and RA 1 to 100 nmol/L inhibited secretion from 83% to 68% of control levels. Inhibition of secretion was dose-dependent. RARa RNA transcripts were detected in αTC-1 and In-R1-G9 total RNA extracts; RARy transcripts were detected in αTC-1 cells. We conclude the following: (1) ROH and RA inhibit glucagon secretion in cultured rat islets and glucagon-secreting cell lines, and in cell lines the effect of RA is dose-dependent; (2) on a molar basis, RA is on the order of 10- to 100-fold more potent than ROH, a finding consistent with RA being the active metabolite of ROH at the c-cell level; and (3) this inhibition may be mediated through classic pathways of retinoid action involving nuclear RARs and gene expression of specific proteins.