The Ly49 family of natural killer (NK) cell receptors are major histocompatibility complex (MHC) class I-specific inhibitory receptors that are distributed to overlapping NK cell subsets. Extending earlier studies of polyclonal NK cell populations, we have employed an analysis of short-term NK cell clones from Ly49A heterozygous mice, to demonstrate that the Ly49A gene is usually expressed from one or the other allele in each Ly49A+ cell. However, we also detected a small percentage of clones that expressed both Ly49A alleles. The possibility that the colonies exhibiting bi-allelic Ly49A gene expression had been inoculated with more than one cell was ruled out by parallel analysis of clones isolated from mixtures of NK cells from Ly49A homozygous mice. The frequency of bi-allelic Ly49A+ clones suggested that the two Ly49A alleles in an NK cell are chosen for expression independently. These data are consistent with the proposal that mono-allelic Ly49A gene expression may arise as a consequence of a stochastic Ly49 gene activation mechanism. Analysis of Ly49A+ clones from MHC-different mice demonstrated that class I-deficient mice harbored a greater number of bi-allelic Ly49A+ cells than did H-2d mice, which express a Ly49A ligand. Although the numbers were insufficiently large for a clear assignment, H-2b mice may harbor an intermediate number of bi-allelic Ly49A+ NK cells. The effects of MHC expression on the prevalence of bi-allelic Ly49A+ cells suggest that an MHC-dependent education process modifies the Ly49 repertoire.