The signaling pathways involved in the regulation of glucocorticoid on Pi uptake were examined in primary cultured rabbit renal proximal tubule cells (PTCs). Dexamethasone (DEX, 10<sup>–9</sup> M) inhibited Pi uptake, although aldosterone, a mineralocorticoid, did not affect Pi uptake. Its effect was due to a 23% decrease in the V<sub>max</sub> value. DEX–induced inhibition of Pi uptake was prevented by actinomycin D, cycloheximide, and the glucocorticoid receptor antagonists, progesterone and cortexolone. SQ 22536 (adenylate cyclase inhibitor) and the myristoylated protein kinase A inhibitor amide 14–22 (PKI) did not block the DEX–induced inhibition of Pi uptake. Indeed, DEX did not affect cAMP production. However, neomycin and U 73122 (PLC inhibitors), staurosporine and bisindolylmaleimide I (PKC inhibitors) blocked the DEX–induced inhibition of Pi uptake. In addition, DEX increased the membrane–bound PKC activity from 2.82±0.21 to 4.16±0.34 pmol/mg protein/min. These findings demonstrate that glucocorticoid inhibits Pi uptake and its effect is genomic and receptor–mediated and the activation of the PLC/PKC pathway is involved in its effect on the PTCs.