Pancreatic triacylglycerol lipase (PTL) is expressed in novel locations during hibernation in the thirteen-lined ground squirrel (Spermophilus tridecemlineatus). PTL cDNAs isolated from two of these locations, heart and white adipose tissue (WAT), contain divergent 5'-untranslated regions (5'-UTRs) suggesting alternative promoter usage or the possibility of multiple PTL genes in the ground squirrel genome. In addition, cDNAs isolated from WAT contain tracts of retroviral sequence in their 5'-UTRs. Our examination of PTL genomic clones isolated from a thirteen-lined ground squirrel genomic DNA library, coupled with genomic Southern blot analysis, enabled us to conclude that PTL mRNAs expressed in heart and WAT are the products of the same single-copy gene. The 5' portion of this gene spans 9.2 kb, is composed of 6 exons, and contains a full-length endogenous retroviral genome with conserved long terminal repeats (LTRs). Alignment of the ground squirrel PTL gene with the mouse, rat, and human PTL genes indicates that this retrovirus inserted into the ground squirrel genome approximately 200 bases upstream of the original PTL transcriptional start site. The insertion is a relatively recent event based on largely intact open-reading frames containing minimal frame-shift and nonsense mutations. The high-percentage identity (99.2%) shared between the 5'- and 3'-LTRs of this endogenous retrovirus suggests that the insertion occurred as recently as 300,000 years ago.