Intestinal calcium (Ca) hyperabsorption is a well-documented feature of experimental phosphorus depletion (PD). To further evaluate the effect of PD on Ca absorption we studied metabolic balance and in vitro everted duodenal sac uptake of Ca and phosphorus (P) in weanling male rats. Animals were assigned to three dietary groups: normal, 0.3% P ad libitum (NP); low, 0.03% P ad libitum (LP); and normal, 0.3% P but pair-fed with assigned LP mates (NP-PF). Results indicate that although PD led to an early but unsustained increase in 45Ca uptake by the everted duodenal sac in vitro, net intestinal Ca retention is consistently decreased in rats on the LP diet compared with rats eating either the NP or NP-PF diet. The reduction in net intestinal Ca absorption is reflected by an increase in fecal Ca, both in absolute quantities and in proportion to dietary Ca intake. The initial negative P balance after the initiation of the LP diet was promptly, albeit precariously, corrected. This was associated with a sustained increase in duodenal 32P uptake in vitro and virtual cessation of growth. Because the biosynthesis of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its accumulation in intestinal mucosa have been reported to increase with PD, our study represents an example in which the physiological interrelationship between the activity of 1,25(OH)2D3 and intestinal Ca absorption may be dissociated.