Insertional mutagenesis represents an inherent risk in retrovirally mediated gene therapy, but it may be a useful experimental strategy for identification and isolation of novel cellular loci. In this investigation we have established a model system using a heterozygous thymidine kinase (tk) marker locus in a human B lymphoblastoid cell line, and a M-MuLV based shuttle vector. The frequency of TK- mutants in cells carrying 1-2 proviruses per genome is approximately 2 x 10(-5), a 5-fold increase as compared to an uninfected control population. Southern analysis of a set of 13 retrovirus infected TK- mutants revealed a predominance of rearrangements among those mutants which had not undergone loss of heterozygosity. No consistent relationship was found to exist between the occurrence of a rearrangement and tk gene expression as detected by northern analysis. The mechanisms of retroviral shuttle vector insertional mutagenesis were characterized in more detail by focusing on a single TK- mutant, T2. The single proviral insert in T2 was found to lie within tk intron 2, in parallel orientation to the direction of tk transcription. DNA sequence analysis of tk cDNA revealed the presence of an aberrantly spliced product from which exon 4 is excluded. Aberrant splicing could sufficiently account for the low level of functional tk transcript and thus the TK- phenotype in T2, although potential contributions from other mechanisms cannot be excluded.