A single intraperitoneal injection of amiloride in the range of 2.7, 5.4, 10.9, and 21 mumol/100 g body wt in female adult rats produced, in the two successive periods of 4 h following its administration, a significant decrease in the urinary excretory rate of kallikrein. Amiloride, 10.9 mumol/100 g body wt, which significantly reduced active kallikrein, also decreased, but less significantly, the trypsin-activated kallikrein in the urine. The fall in the excretory rate of kallikrein cannot be explained by its enzymatic inhibition by amiloride, since the inhibition was only present at higher concentrations. In hyperhydrated rats amiloride did not change the kallikrein excretory rate in the urine collected within 4 h after the injection. Rats simultaneously injected with 7.6 mumol/100 g body wt furosemide and 10.9 mumol/100 g body wt amiloride excreted levels of kallikrein similar to those found in rats injected with furosemide alone. The kidneys of rats removed after 4 h of administration of 10.9 mumol/100 g body wt amiloride showed a significant lowering of the kallikrein activity compared with the respective controls. The decrease of renal kallikrein tended to be similarly pronounced in those rats that received amiloride and furosemide simultaneously. These results confirm the depressive effect of amiloride on kallikrein excretion, which may be explained by an inhibitory action on kallikrein release, activation, and synthesis by the renal cells.