The in vitro methylation of three liver cytosolic proteins was found to be selectively stimulated by calmodulin. This effect was also seen, although to a much smaller degree, in kidney and lung, but not in testes, brain, or spleen. The three methylated proteins affected by calmodulin have apparent Mr = 29,000, 32,000, and 45,000. The stimulation of methylation by calmodulin was greatest for the Mr 29,000 protein; there was an equal degree of methylation of the other two proteins. Dialysis of liver cytosolic fractions also stimulated the methylation of these proteins; the methylation of the Mr 32,000 and 45,000 proteins was stimulated to a greater extent by dialysis than by calmodulin. The degree of stimulation of methylation of the Mr 29,000 protein by calmodulin and dialysis was equivalent, but the addition of calmodulin to dialyzed liver cytosolic fractions gave additive effects on the stimulation of methylation of the Mr 29,000 protein, but not of either the Mr 32,000 or 45,000 proteins. Troponin C stimulated the methylation of the Mr 29,000 protein, but not the Mr 32,000 or 45,000 proteins, whereas parvalbumin stimulated methylation of the Mr 32,000 protein, but not the Mr 29,000 or 45,000 proteins. The effects of calmodulin and dialysis on protein methylation are cation-dependent and substrate-specific; methylation of the Mr 29,000 was supported by Mn2+, Ca2+, and Co2+, and to a lesser degree by Mg2+, Ni2+, and Zn2+. Methylation of the Mr 32,000 protein was supported only by Mn2+ and Mg2+ and methylation of the Mr 45,000 protein by Mn2+, Mg2+, Ca2+, Ni2+, and Zn2+, and to a much smaller extent by Fe2+. In extracts of fetal liver, stimulation of protein methylation by calmodulin or dialysis was restricted to the Mr 45,000 protein. In regenerating liver, stimulation of the methylation of all three proteins was observed, but the stimulation provided by dialysis plus calmodulin was much less than that observed in preparations from intact adult liver, suggesting a possible negative correlation between the rate of cell division and calmodulin-dependent methylation of these hepatic proteins. These results are consistent with the presence in liver of a minimum of three distinct N-methyltransferases and a dialyzable inhibitor which antagonizes calmodulin-dependent protein methylation.