Monolayers of opossum kidney (OK) cells are widely used as models for the renal proximal tubule. OK cells adapt to phosphate (Pi) depletion by increasing their capacity for apical and basolateral Na<sup>+</sup>-dependent Pi uptake. Because NMR-visible cell Pi was found to be decreased in Pi-deprived kidney cells, we suggested that up-regulation of basolateral Pi efflux also occurs during adaptation to Pi deprivation [ American Journal of Physiol 1994;267:C915–919]. In order to test this hypothesis, we measured the cell Pi pool, basolateral Pi efflux and transepithelial Pi fluxes in OK cells grown on permeable plastic filters, exposed overnight to solutions containing either 0.5 m M (deprived) or 2.0 m M (replete) Pi or <sup>32</sup>Pi. Following steady state or acute loading with <sup>32</sup>Pi, the specific activity (SA) of cell Pi, the cell Pi pool and the basolateral efflux of <sup>32</sup>Pi were measured. In the steady state, a 2-fold increase in Pi uptake sustained the intracellular Pi pool at 85% of the control level (30 ± 5 nmol/mg) in spite of a decrease in extracellular Pi from 2 to 0.5 m M. When the extracellular Pi was acutely (1 h) reduced to 0.1 m M, the cell Pi pool decreased (to 3 ± 1 nmol/mg) both in cells previously adapted overnight to either 0.5 or to 2 m M Pi (p >0.3). The rates of absolute and fractional basolateral washout of cell <sup>32</sup>Pi after 1 h loading with 0.1 m M<sup>32</sup>Pi were similar in cells adapted to 0.5 compared to 2 m M Pi. This indicates that Pi depletion did not affect the effective permeability of the basolateral membranes to Pi. Adaptation for 16 h to 0.5 compared to 2 m M Pi did not alter the rate of net transepithelial transport of 0.1 m M Pi from the apical to the basal compartment but reduced (p < 0.05) the unidirectional fluxes of both <sup>32</sup>Pi and <sup>14</sup>C-mannitol. An insufficient driving force (unchanged or low Pi concentration in the transport pool, low electrical or coupled-anion gradients) and a constant effective basolateral Pi permeability must have limited basolateral Pi efflux in cells exposed to 0.1 m M Pi. Thus, in OK cells grown on plastic support there are no adaptive increases in either basolateral Pi efflux, or in transcellular and paracellular Pi transport, in response to Pi depletion. Adaptations are limited to increases in apical and basolateral sodium-dependent Pi uptakes that can maintain the cell Pi pool as long as apical Pi is not too low (≧0.5 m M). The OK cells adapt to low Pi concentrations conserving cell Pi but not increasing basolateral Pi efflux nor transepithelial Pi transport.