The T-cell cloning assay detecting mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus provides a well developed system for studying human somatic gene mutation. The hprt mutational spectrum comprises missense, nonsense and splice mutations, as well as large structural alterations including deletions, duplications and insertions. Only few of the hprt deletions, which represent 10-15% of background in vivo mutations in T-cells of adults, have been characterized in detail at the genomic level, and the mechanisms involved in the majority of hprt structural alterations remain unknown. Illegitimate activity of V(D)J recombinase resulting in deletion of hprt exons 2 + 3 has been shown to account for 40% of the hprt mutations in T-lymphocytes of human newborns and a few percent of the mutations in adults. In this report, novel recombinational mechanisms were identified by characterization of two T-cell mutants. One mutant derived from a healthy adult was found to have a 3.2-kb genomic insertion in the first intron of the hprt gene, and a 369-bp T-cell receptort (TCR) alpha gene sequence between exons 1 and 2 of its hprt cDNA. This mutation provides unique and direct evidence for illegitimate recombination between the TCR gene and the hprt gene in human T-lymphocytes in vivo. Moreover, the mutation identifies a novel cDNA sequence for the TCR alpha chain variable region. Another hprt- mutant, obtained from a T-cell culture treated with acetaldehyde, showed that splice mutation can be caused by a large deletion detectable on Southern blot. This 3.4-kb deletion involved both intron 1 and exon 2 sequences and was flanked by 5-bp direct repeats. The utilization of a novel cryptic acceptor site in intron 1, located far upstream from the lost consensus splice site, resulted in a partial inclusion of the intron 1 sequence in the hprt cDNA.