Activin A stimulates insulin secretion in pancreatic β-cells by a calcium-dependent mechanism. The present study was conducted to further characterize the effects of activin A in two glucose-responsive insulinoma cell lines, MIN6 and HIT-T15 cells. In HIT-T15 cells, activin A evoked an increase in cytoplasmic free calcium concentration, stimulated insulin secretion, maintained glucose responsiveness of the cells and inhibited DNA synthesis. However, activin A did not have any effect in MIN6 cells. Measurement of 125I-labeled activin A binding in MIN6 cells revealed that the number of binding sites was markedly reduced, suggesting that the refractoriness was due, at least partly, to the reduced numbers of the activin receptor. Stable transfectants of MIN6 cells that overexpressed the type II activin receptor were then developed. The transfected cells (MIN6-ActR cells) expressed ten times more 125I-labeled activin A-binding sites than parental cells and the apparent Kd was 1·15 nm, which was nearly identical to that in parental cells. Affinity cross-linking in MIN6-ActR cells showed that a 90 kDa type II receptor as well as a 52 kDa protein, presumably follistatin, was markedly labeled with 125I-labeled activin A. Although MIN6-ActR cells expressed significant numbers of activin receptors, activin A did not induce immediate calcium-dependent responses in these cells. In contrast, activin A was capable of inducing long-term effects in MIN6-ActR cells; thus, reduction of the glucose concentration in culture medium from 25 to 5·5 mm for 4 days resulted in a remarkable loss of insulin response to glucose stimulation but this decline in response to glucose was prevented by the addition of activin A during culture. In addition, activin A inhibited DNA synthesis in MIN6-ActR cells. Hence, although activin A did not induce calcium-dependent responses, it evoked some calcium-independent effects in MIN6-ActR cells. Taken together, activin A elicits various effects in β-cells by both calcium-dependent and -independent mechanisms.