Although rapid-onset, short-term regulation of neuronal Ca currents by neurotransmitters and second messengers is well documented, little is known about conditions that can cause longer-lasting changes in Ca channel function. We report here that persistent depolarization is accompanied by slowly developing long-term reduction of neuronal Ca currents. Rat myenteric neurons grown in cell culture for 1–7 d were studied with the tight-seal whole-cell recording technique. Macroscopic Ca-channel currents had decaying and sustained components at all days studied. When the neurons were grown in medium containing 25 mM KCl, which depolarized them to -40 mV and caused significant elevation of intracellular Ca, the densities of both components of Ca-channel current decreased by 40–80%. Several results suggest that different mechanisms underlie the downregulation of the two components. (1) The density of the decaying component decreased approximately four times faster than did that of the sustained component. (2) When neurons were returned to control medium, which contained 5 mM KCl, the density of the sustained component returned to control levels within 24 hr, while that of the decaying component did not recover significantly. (3) Inhibitors of RNA and protein synthesis reduced or prevented downregulation of the sustained but not of the decaying component. (4) The dihydropyridine antagonist nitrendipine, which prevented the sustained elevation of intracellular Ca in neurons grown in 25 mM KCl, prevented downregulation of the sustained component but had no effect on downregulation of the decaying component. We suggest that these forms of regulation of Ca current density could help neurons adapt to altered levels of electrical activity and may contribute to changes in synaptic strength that occur during periods of increased or decreased electrical activity.