Gluconeogenesis is a major metabolic function of the renal proximal tubular cell. To characterize the regulation of this process in proximal tubule, glucose production from gluconeogenic precursors was measured in proximal tubular segments prepared from dog kidney. Production of glucose was a linear function of time for up to 120 min of incubation at 37 degrees C under a variety of conditions. Lowering the pH of incubation media from 7.5 to 7.0 increased glucose synthesis. Production of glucose was inhibited by 3-mercaptopicolinate. Incubation of proximal tubular segments with insulin diminished synthesis of glucose. Incubation of segments with tumor-promoting phorbol esters, 12-O-tetradecanoylphorbol-13-acetate or phorbol 12,13-dibutyrate, resulted in decreased production of glucose. This effect was not observed following incubation with the inactive phorbol ester 4 alpha-phorbol. Changes in glucose synthesis could not be attributed to alterations in cell viability or in rates of glucose oxidation induced by experimental maneuvers. Our findings confirm the usefulness of proximal tubular segments for characterization of metabolic processes in this portion of the nephron. The experimental results are consistent with a role for protein kinase C in the control of gluconeogenesis in proximal tubule.