Down-regulation of c-myb mRNA levels by [Ca2+]i-increasing agents (A23187, thapsigargin, cyclopiazonic acid) and erythropoietin was comparatively studied in the erythropoietin-responsive murine erythroleukemia cell line, ELM-I-1. The Ca2+-induced suppression of c-myb mRNA could be inhibited by the calmodulin antagonists trifluoperazine and calmidazolium, as well as by cyclosporin A, an inhibitor of the Ca2+/calmodulin-dependent protein phosphatase 2B (calcineurin). KN-62, an inhibitor of Ca2+/calmodulin-dependent protein kinases, did not antagonize the Ca2+-mediated decrease in c-myb mRNA. In cyclosporin A-treated ELM-I-1 cells, a close correlation could be demonstrated between the antagonization of the Ca2+ effect on c-myb mRNA levels and inhibition of the calcineurin phophatase activity. On the other hand, FK506, which did not inhibit calcineurin activity in ELM-I-1 cells, failed to prevent the Ca2+-mediated decrease in c-myb mRNA. The erythropoietin-induced down-regulation of c-myb mRNA levels could be demonstrated also in the presence of EGTA and was resistant to calmodulin antagonists and cyclosporin A. In addition, no increase in [Ca2+]i was observed in ELM-I-1 cells in response to erythropoietin. Cyclosporin A inhibited the Ca2+-induced hemoglobin production, while the erythropoietin-mediated increase in hemoglobin synthesis was not affected. The results indicate that the Ca2+-induced decrease in c-myb mRNA and increase in hemoglobin synthesis is mediated by calcineurin, while these effects of erythropoietin occur independently of Ca2+ in ELM-I-1 cells. Calcineurin may be involved in the regulation of c-myb expression in erythroid precursor cells and Ca2+ signals via calcineurin may positively modulate the differentiation inducing action of erythropoietin.