We utilized a strain of mice, derived from a radiation mutagenesis experiment and carrying an activity-attenuated allele of the X-linked enzyme glucose-6-phosphate dehydrogenase (G6PD), to analyze the development of the cell lineage leading to cerebellar Purkinje neurons. Due to random X inactivation during early embryonic development, X- linked genes can be used to distinguish between clonally related populations of cells in X inactivation mosaics. Following histochemical staining for G6PD activity, the numeric proportions of Purkinje cells expressing either the wild-type or the mutant enzyme and the spatial distribution of these cellular phenotypes and their relation to anatomically and genetically defined cerebellar compartments were analyzed. Our data suggest that cerebellar Purkinje neurons originate from a limited pool of some 129 precursors. The size of this pool is different from the one derived from chimeric mice, allowing us to deduce the relative timing of Purkinje cell lineage restriction. Our data also show that Purkinje neurons of distinct lineage are extensively intermingled within the cerebellar cortex. Together, these findings suggest both a role for cell-cell communication in the development of genetically defined cerebellar compartments and a temporal window during which such cellular interactions may take place.