The hprt clonal assay detects mutations occurring in vivo in the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene of human T-lymphocytes. Analysis of 94 wild-type and 326 hprt mutant clones from 3 normal males was performed using Southern blotting with hprt and T-cell receptor (TCR) gene probes. Gross structural alterations of the hprt gene occurred in approximately 14% of the in vivo derived mutants. Breakpoints were randomly distributed across the gene with one possible mutational "hot spot" observed. Most hprt mutants were independent as judged by TCR gene rearrangement patterns indicating that the measured hprt mutant frequency is a good measure of the actual hprt mutation frequency. However, sibling mutants (generally doublets and triplets except for one nonamer) were detected. Information on the timing in vivo of the hprt mutational events and the persistence in vivo of sibling mutants was also obtained.