The renal handling of immunoreactive insulin was studied in the isolated perfused normothermic rat kidney to determine (a) the relative contributions of glomerular clearance and peritubular clearance to the renal clearance of insulin under different conditions, (b) what metabolic factors influence the ability of tubular cells to remove insulin from the glomerular filtrate and the peritubular circulation, and (c) whether the same factors influence the luminal and contraluminal uptake of insulin.In control kidneys the organ clearance of insulin (OCi) was 974+/-63 mul/min (SEM), of which a maximum of 46% could theoretically be accounted for by filtration. OCi was not altered by fasting, lack of exogenous fuel (glucose), or the addition of cyanide. The glomerular filtration rate did not correlate with the OCi, but there was a significant (P < 0.001) negative correlation (r = -0.828) between the peritubular clearance and glomerular filtration rate. Both N-ethylmaleimide and cold (10 degrees C) reduced the rate of insulin removal. Fractional excretion of filtered insulin (9.7+/-1.7% in controls) was not significantly altered by fasting or perfusing without glucose. In contrast, KCN increased fractional excretion of insulin to 41.9+/-3.7% whereas cold increased fractional excretion to 69.0+/-3.3%. This study indicates that renal tubular cells remove insulin from the tubular lumen and the peritubular compartment. Furthermore, the data suggest that insulin removal by tubular cells is a temperature-sensitive process consisting of two different systems. The system associated with the luminal aspect of the cell appears to be dependent on oxidative metabolism, whereas the system associated with the contraluminal aspects of the cell appears to be independent thereof. Under several circumstances when the glomerular clearance of insulin falls thereby reducing the amount of insulin absorbed by the luminal aspect of the cell, contraluminal uptake increases, and a constant rate of insulin removal is maintained by the kidney.