Irradiation of DNA in a cellular environment leads to many types of DNA damage, resulting from various effects of gamma-radiation. One of these effects is the formation of water-derived radicals (e.g., .OH radicals), which are formed in the vicinity of DNA (indirect effect). To study the influence of the indirect effect on gamma-radiation-induced mutations, a newly constructed plasmid, containing the lacI gene as a target gene, was irradiated with 60Co gamma-radiation in aqueous solution, in the presence of oxygen. Under these circumstances, only .OH radicals will be responsible for the induced mutations. Sequence analysis of the gamma-radiation-induced mutations showed that 96% of all mutations were base pair substitutions, 87% of which occurred in the lacI gene, the others are formed in the lac operator part. All gamma-radiation-induced mutations in the lacI gene occurred exclusively on G:C base pairs, and no mutations at A:T base pairs could be detected. In the spontaneous mutation spectrum, 83% of all mutations were base pair substitutions, 35% of which occurred in the lacI gene and 48% in the lac operator part. Base pair substitutions on G:C base pairs were very similar in the gamma-radiation-induced and in the spontaneous mutation spectrum, implying a high contribution of .OH radicals to spontaneous mutagenesis. A:T to G:C transitions accounted for 10% of all spontaneous base pair substitutions in the lacI gene and are probably the result of effects, other than just .OH radicals. It can be concluded that .OH radicals are an important source for mutations at G:C base pairs. In this paper, the extracellular gamma-radiation-induced mutation spectrum is also compared to the previously obtained, intracellular gamma-radiation-induced mutation spectrum of the lacI gene. Comparison shows some differences, such as relative high amounts of mutations at A:T base pairs, G:C to T:A transversions and frameshift mutations in the intracellular gamma-radiation-induced mutation spectrum, as compared to the extracellular gamma-radiation-induced mutation spectrum. Since the extracellular gamma-radiation-induced mutation spectrum shows that .OH radicals are mainly responsible for base pair substitutions on G:C base pairs, mutations at A:T base pairs in the intracellular gamma-radiation-induced mutation spectrum are apparently the result of additional or other factors.