Chronic dietary phosphate restriction is associated with up-regulation of sodium-dependent phosphate (Na/P(i)) cotransport by renal proximal tubular epithelial cells in association with increases in Na/P(i) cotransporter mRNA and protein. We investigated whether changes in cytosolic calcium mediate this adaptive response in opossum kidney cells, a continuous line of renal epithelial cells. After 24 h of phosphate depletion, steady-state cytosolic calcium levels were increased; this increase was observed at physiologic levels of phosphate restriction and was prevented by the calcium channel blocker verapamil. Chronic phosphate depletion was also associated with parallel increases in Na/P(i) cotransport activity, Na/P(i) cotransporter mRNA, and NA/P(i) cotransporter protein, all of which were blocked in verapamil-treated cells. Actinomycin D, at a dose that prevented the increase in NaPi-4 mRNA during phosphate depletion, also prevented the increase in Na/P(i) cotransport activity. Incubation with the calcium ionophore ionomycin or A23187 reproduced the increase in Na/P(i) cotransporter mRNA in phosphate- replete cells. Conversely, chelation of cytosolic calcium by quin-2/AM prevented the increase in Na/P(i) cotransporter mRNA in phosphate-depleted cells. The effect of an increase in cytosolic calcium was specific for the Na/P(i) cotransporter as mRNA levels for the sodium-dependent glucose transporter were not affected. Our observations suggest that chronic phosphate restriction increases steady-state cytosolic calcium, which, in turn, increases transcription of Na/P(i) cotransporter mRNA, thereby stimulating Na/P(i) cotransport activity.