Addition of Ca2+ to primary cultures of female pituitary cells incubated in serum-free medium lacking added Ca2+ yielded no effects on levels of prolactin or growth hormone mRNA, assayed by cytoplasmic dot hybridization. However, incubation of the cells in serum-free medium containing sufficient ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to reduce medium Ca2+ levels below the 10-40 microM present as a trace contaminant yielded a decrease in the levels of both mRNAs. The decrease was dose-dependent at extracellular Ca2+ concentrations below 1.0 microM, had an apparent half-maximum at about 0.3 microM, and did not appear to plateau with increasing incubation times. Following 2-3-day incubations of cells in low Ca2+, a reduction of prolactin mRNA (23-70-fold) consistently greater than the reduction of growth hormone mRNA (9-15-fold) was observed. Similar effects of reduced extracellular Ca2+ were obtained with primary cultures of male pituitary cells. The specificity of these effects of lowered extracellular Ca2+ was demonstrated by the following observations. The decreases in these mRNAs were substantially reversible by readdition of Ca2+ to the incubation medium. Reduction of extracellular Ca2+ led to no detectable changes in cellular ribosomal RNA levels or over-all RNA synthesis. In male pituitary cells, the level of another metal-regulated mRNA, that for metallothionein, was not decreased by a reduction of extracellular Ca2+ that caused a 40-fold decrease in levels of prolactin and growth hormone mRNA. Hence, Ca2+ exhibits specificity in its regulation of pituitary prolactin and growth hormone gene expression.