A search for mitotic recombination was carried out using mutant subclones of cultured Chinese hamster ovary cells. Recombination events were sought between the linked loci specifying the enzymes hypoxanthine phosphoribosyl transferase and glucose-6-phosphate dehydrogenase. It was shown by fluctuation analysis that markers at these two loci co-segregate from doubly heterozygous pseudotetraploid hybrid cells more than 90% of the time. The minority class of segregants, which had lost one marker without losing the other, were genetically analyzed to distinguish between the possibilities of mitotic recombination and deletion of chromosomal material. Nine clones in which a linkage disruption had occured were studied, using further cell hybridization and segregation. In three cases, a recessive lethal loss of genetic information was indicated, suggesting the deletion mechanism. In six cases, it was demonstrated that no new linkage relationships had been established concomitant with linkage disruption. Thus, in all nine clones, the evidence indicated that mitotic recombination was not involved in the events that disrupted linkage between these two loci. If mitotic recombination takes place at all in this system, the rate must be less than about 10-6per cell per generation.