We have used free-flow micropuncture to study the tubular locus at which kallikrein enters the urine. Kallikrein was measured by a newly developed, very sensitive assay for kininogenase activity; active kallikrein was measured directly by this assay and total kallikrein after activation of inactive kallikrein. Kallikrein was readily detected in all of 17, late distal tubular fluid-samples. In contrast, kallikrein was too low to detect in 15 of 17 proximal or in 11 of 14 early distal tubular fluid samples. Calculations indicate that less than 10% of urinary kallikrein could have derived from filtration or from proximal secretion of kallikrein. We conclude that urinary kallikrein enters the urine via secretion in the distal tubule. Filtration or proximal secretion of kallikrein does not contribute significantly to urinary kallikrein excretion.