1. We have cloned and characterized a new species of kallikrein cDNA from a human colon cDNA library. The new kallikrein cDNA clone contains a part of intron 2 of the tissue kallikrein gene which is spliced to the remaining exon sequences. It does not contain exons 1 and 2. 2. An in-frame open reading frame is present in the new kallikrein cDNA allowing translation of a 216-amino acid product. The intron-containing kallikrein transcript was detected in salivary glands, pancreas, kidney, colon, prostate gland, testis, spleen, and lung by reverse-transcription/polymerase chain reaction followed by Southern blot analysis using an intron-containing kallikrein-specific oligonucleotide probe. 3. The results indicate that the new species of kallikrein may be processed by alternative splicing or arises from a different transcription initiation site.