A hybrid cell line (Hy3-5) derived from BHK21/13 hamster fibroblasts was tested for the occurrence of segregants. The hybrid line was obtained by co-cultivating in the presence of aminopterin cells deficient in inosinic acid pyrophosphorylase and cells deficient in thymidine kinase. These enzymatic deficiencies conferred upon these cells resistance to either 6-thioguanine (TG) or 5-bromodeoxyuridine (BUDR). As expected, the hybrid line was sensitive to both analogues. Survival curves to TG and BUDR of Hy3-5 cells show the presence of resistant fractions, which are absent in several non-hybrid clones derived from BHK21/13. The resistant cells have reduced chromosome numbers; the mean is lowest in TG-resistant cells. A sib-selection experiment and a fluctuation test indicate that resistant cells arise spontaneously and represent two independent fractions of the hybrid cell population. The latter conclusion is supported by the lack of cross-resistance displayed by the two cell types. A karyotypic analysis suggests that the loss of chromosomes observed in TG-resistant cells is not random. As a whole, these results support the interpretation that cells of either parental type selected from line Hy3-5 are chromosomal segregants which have lost, in each case, non-overlapping groups of chromosomes. © 1969.