Among the major proteins in the rat seminal vesicle secretion, transglutaminase catalyzed the cross-links among RSVS 1-III. Six peptide sequences determined from the trypsin digests of RSVS III were confirmed in the protein sequence derived from two paralogs, RSVS IIIalpha and RSVS IIIbeta gene, in rat 3q42. Their transcription units are organized with the first exon encoding a signal peptide, and the second a secreted protein, whereas the third encompasses a 3'-non-translated nucleotide that shares common features of rapidly evolving substrates of transglutaminase (REST) gene family. These two genes have 99%, identity ill their coding region and both express in adult rats with the same transglutaminase cross-linking site, manifesting functional preservation. All of REST genes reported thus far for human and Muridae were mapped in a chromosomal locus between KCNS1 and SLPI suggesting the locus as an active evolving region. The molecular evolution of this gene family is discussed. (C) 2004 Elsevier Inc. All rights reserved.