Ca2+ has been proposed to regulate expression of the gene for the Ca2+ pump of the sarcoplasmic reticulum in developing chicken myoblasts (A. N. Martonosi, L. Dux, R. L. Terjung, and D. Roufa. Ann. NY Acad. Sci. 402: 485-514, 1982. In the present study, intracellular free Ca2+ ([Ca2+]i) was measured with the photoprotein, aequorin, incorporated by a reversible hyperpermeabilization technique, and with the fluorescent probe, quin 2. Because aequorin reacts irreversibly with Ca2+ and is inactivated by heat, at 37 degrees C the active aequorin content declined markedly. The resting glow (1/10(6) of the maximum light) disappeared after a few hours, whereas the light from the total active aequorin remaining was still detectable when cells were lysed 3 days later. A new approach, which compared the rate of disappearance of aequorin in treated and control cells, was developed on the basis that aequorin would be inactivated more quickly in cells with higher [Ca2+]i. A23187, ionomycin, and trifluoperazine, all of which accelerated gene expression (A. N. Martonosi et al.), increased the rate of decay of active aequorin and therefore increased [Ca2+]i. "Ca shock", and ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid, which also increased the gene expression (A. N. Martonosi et al.), caused small increases of [Ca2+]i not detectable by aequorin but detectable by quin 2. X537A and ouabain had no effect on the gene expression (A. N. Martonosi et al.) and did not raise [Ca2+]i. The results support the proposal that Ca2+ may regulate expression of this gene.