The primary structure of human kidney kallikrein has been elucidated by molecular cloning and cDNA sequence analysis. Structural homology between mammalian glandular kallikreins permitted the isolation of several clones from a human kidney cDNA library where kallikrein sequences represented approximately 0.01% of the cDNA. Identity of the cloned sequence with the published amino-terminal sequence of human urinary kallikrein strongly implicates the kidney as a major source of this protein. The overall structure of the kidney enzyme is 67% homologous to that of the corresponding mouse kidney kallikrein and only 60-62% homologous to the other published mouse kallikrein sequences. Homology to the pancreatic kallikreins of pig and rat is 67% and 61%, respectively. As expected, the amino acids required for catalytic activity are conserved as is the Asp residue required for the kallikrein-type specificity. Southern blot analysis demonstrates the presence of a number of related sequences in human DNA, although these do not appear to be as numerous as those in the mouse genome. Several polymorphisms in the kallikrein genes were observed.