The human haploid genome contains a family of at least five different transglutaminases that are differentially expressed in time- and tissue-specific ways. Of these, transglutaminase 3 (TGase3) is unusual in that it is a pro-enzyme requiring activation by proteolysis. To date it is known to be expressed only in terminally differentiating epidermal and hair follicle keratinocytes. In this paper we show that it is encoded by a gene (TGM3) of 42.8 kbp containing 13 exons. In the course of isolation of genomic clones for the TGM3 gene, we also found clones encoding the widely expressed tissue or TGase2 enzyme, perhaps due to high degrees of sequence homology. The structure of the TGM2 gene has not yet been reported. Our incomplete data suggest its exon/intron organization is very similar to that of TGM3. Although the common intron splice points of all members of the transglutaminase gene family have been conserved, the TGM3 and TGM2 genes, and the gene for the subplasma membrane transglutaminase-like protein band 4.2, lack two introns found in the TGM1 and factor XIIIa genes, and the exact intron splice point of another intron is shifted with respect to that of the TGM1 and factor XIIIa genes. Based on sequence homologies and gene structures, the data support a phylogenic tree in which the TGM2 and TGM3 genes belong on a branch distinct from other transglutaminases.