The participation of second messenger pathways in 1,25(OH)2D3-induced stimulation of protein synthesis in chick embryo myoblasts undergoing proliferation was studied. Double-labelling experiments with 14C- and 3H-leucine showed the induction by the hormone of proteins with apparent molecular masses (treatment interval) of 60 kDa (1 to 2 h), 70 kDa (2 to 4 h), 80 kDa (4 h) and a 19 kDa protein (6 to12 h) previously identified as calmodulin. The PKC activator TPA and the Ca2+ ionophore X-537 A did not mimick the effects of the sterol on protein synthesis whereas similar double-labelling patterns were obtained with forskolin, an adenylate cyclase activator. Dot-blot and Northern hybridization analysis revealed increased calmodulin mRNA levels in response to both the hormone and forskolin. These results involve the cAMP messenger system in 1,25(OH)2D3 stimulation of calmodulin synthesis and may be relevant to understand hormone regulation of muscle cell proliferation. © 1993 Academic Press. All rights reserved.