The present studies were performed to investigate the kinetics of regulation of water channels and urea carriers in the rat terminal (IMCD) in response to vasopressin (AVP). The time courses of osmotic water permeability (Pf) and urea permeability (P(urea)) were measured in isolated perfused rat terminal IMCD segments following AVP stimulation and subsequently following AVP washout. Under control conditions, Pf and P(urea) kinetics were similar. Both transport processes exhibited complex patterns of activation with a period of rapid permeability increase followed by a period of slower increase. Both transporters also exhibited complex patterns of reversal following AVP washout, with a rapid permeability decrease (5 min) followed by a slower decrease toward the baseline value. The measurements were repeated in the presence of a lumen > bath osmotic gradient, a condition associated with a decreased rate of apical endocytosis in collecting ducts. The lumen > bath gradient did not alter the kinetics of Pf increase after AVP addition, but completely blocked the decrease in Pf normally seen with washout of AVP. In contrast, the lumen > bath osmotic gradient did not affect the decrease in urea permeability after AVP washout, but blocked the rapid phase of urea permeability increase following AVP addition. Thus imposition of a lumen > bath osmotic gradient resulted in separation of the time courses of P(urea) and Pf changes associated with AVP addition and washout. This finding indicates that the physical processes responsible for AVP-mediated alteration of urea transporter and water channel activity in the apical membrane are distinct.