Definitive evidence for the occurrence of cell fusion in tumorigenesis was sought in methylcholanthrene-induced sarcomas. This was approached by using allophenic mice generated from strains differing for electrophoretic variants of the ubiquitous, dimeric enzyme glucose phosphate isomerase, with fusion assessed by heterodimer formation. Eight-three carefully trimmed primary tumor samples (from 23 individual tumors in allophenic mice) were analyzed, as were 1,140 clones derived from them. In all primary tumor samples, zymograms exhibited one GPI homopolymeric band. Expression of a hybrid band (indicative of a fusion event) was not observed in these samples. However, 9 (0.8%) of the tumor clones demonstrated a distinct and reproducible hybrid band which was uniformly lost upon recloning. Our data suggest that cell fusion, although uncommon, occurs in the clonogenic cell fraction during primary MCA tumorigenesis and is followed rapidly by chromosome segregation.