To gain direct insight into the action of the second messenger Ca2+ on transcriptional regulation, we have developed an intact cell model in which the intracellular free Ca2+ concentration ([Ca2+]i) can be measured, set, and varied at any level within the physiological range and in which the expression of early response genes is assayed in parallel. Using promyelocytic HL-60 cells, we have observed an exquisite sensitivity to Ca2+ of c-fos, c-jun, and zif268 mRNA accumulation, since early and maximal inductions were observed at 200-300 nM [Ca2+]i. At early times (10-20 min), the [Ca2+]i dose dependence of c-fos transcription and mRNA accumulation displayed a bell shape since c-fos expression was barely modified at high (700-1,250 nM) [Ca2+]i. The threshold [Ca2+]i concentration for prolonged (60 min) c-fos mRNA accumulation was greater than 200 nM. This indicates that the quantitative effects of Ca2+ on a given gene can vary markedly as a function of both the [Ca2+]i concentration and the duration of stimulation. Strikingly, a [Ca2+]i perturbation of only 1 min was sufficient for full induction of c-fos and zif268 transcripts. This demonstrates that a transient perturbation of [Ca2+]i has long term effects on gene expression. The half-life of c-fos mRNA (16 min) was unaltered by Ca2+. Nuclear run-on analysis of the distribution of RNA polymerase II along the c-fos locus indicated that Ca2+ promotes a small increase in transcriptional initiation and a pronounced relief of a block to transcriptional elongation beyond intron 1. The extreme sensitivity to [Ca2+]i, in terms of both the length of time and the dose of [Ca2+]i required for maximal gene induction, demonstrates that Ca2+ is a major physiological regulator of early response gene expression. In addition, the results indicate that a c-fos intragenic element is the main target of Ca(2+)-regulated transcriptional activation.