Loss of heterozygosity (LOH) is an important event in tumor formation. We have used polymorphic microsatellite repeat markers to identify and characterize LOH in spontaneous mutants of a human cell line, MR12-1, that is heterozygous for the adenine phosphoribosyltransferase gene (APRT+/-) located on chromosome 16q24.3. Initially, clones without extensive LOH (which are likely derived as a consequence of intragenic point mutations) and clones with multilocus LOH (which are likely due to major chromosome alterations) were identified. Clones with major regions of LOH were further characterized by assaying additional informative microsatellite markers. Analysis of 20 spontaneously-arising, independent APRT-/- clones from MR12-1 demonstrated that nine of the mutants retained both copies of APRT and 11 had undergone multilocus genetic alterations. The nature of LOH in four of the latter clones has been examined in detail by karyotype and fluorescence in situ hybridization analysis (Shao et al., 1996). These data demonstrate that LOH of chromosome 16 may be due to mitotic recombination, interstitial or partial deletion, or to more complex mechanisms. LOH in these clones may be a consequence of events similar to those observed in many tumors.