Elevated blood ionized Ca concentration appears to be necessary for suppression by chronic metabolic acidosis (CMA) of serum 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] levels in vitamin D-replete rats eating a low-Ca diet (LCD). The present study asks whether in vitro 1,25(OH)2D3 production by proximal tubule cells from such rats is suppressed and whether suppression can be reversed in vitro by an altered ionic milieu. Young rats were fed LCD and given 1.5% NH4Cl in their drinking water for 10 days to produce CMA. Compared with controls, CMA rats had low serum 1,25(OH)2D3 levels. Tubules prepared from CMA rats produced 1,25(OH)2D3 at a low rate compared with control; variation of medium Ca and pH from 7.2 to 7.4 did not increase 1,25(OH)2D3 production. Reducing medium phosphorus concentration ([P]) increased 1,25(OH)2D3 production by tubules from control but not CMA rats. Increasing medium [P] increased 1,25(OH)2D3 production by tubules from CMA but not control rats. CMA appears to alter proximal tubule cell response to medium [P] so that 1,25(OH)2D3 production rises when medium [P] is increased but not when it is reduced. Medium pH and Ca concentration do not appear to be important regulators of renal 1,25(OH)2D3 production in short-term incubations.