In OK cells and in kidney proximal tubules, Pi depletion increases apical (A) and basolateral (B) Na⁺-dependent Pi influxes. In OK cells' monolayers in contrast to proximal tubules, trans-epithelial Pi transport does not increase. This limitation may be due to altered cell-matrix interactions. A and B cell ³²Pi uptakes, and transepithelial ³²Pi and 14C mannitol fluxes were measured in OK cells grown on uncoated or Matrigel-coated filters. Cells were exposed to solution of low (0.25 mM) or high (2.5 mM) Pi. When grown on Matrigel, inmunofluorescence of apical NaPi4 transporters increased, A and B ³²Pi uptakes into Pi depleted cells were 5 and 3-fold higher than in Pi replete cells (P<0.001). Pi deprivation increased A to B (4.6 x, P<0.001) more than B to A (3.5 x, P<0.001) Pi flux and net A to B Pi transport increased 10-fold (P<0.001). With Pi depletion B to A (3.4x) and A to B (3.3x) paracellular ¹⁴C mannitol fluxes increased similarly and its net flux was opposite to that of Pi. In cells grown on uncoated filters, transepithelial and paracellular Pi fluxes decreased or did not change with Pi depletion, despite 2-fold increases in A and B Pi cell influxes. In summary, Matrigel-cell interactions, particularly in Pi depleted cells, led to enhanced expression of apical NaPi4 transporters, higher Pi transport rates across cell boundaries, apical Pi readily entered the transcellular transport pool and paracellular fluxes were smaller fractions of transepithelial Pi fluxes, leading to increased net transepithelial apical to basolateral Pi transport.