Understanding the significance of somatic mutations requires knowledge of the mutations that occur in vivo in healthy people. The molecular characterization of mutations in the hypoxanthine phosphoribosyltransferase (hprt) gene in 217 independent T-lymphocyte mutants from 172 donors, including smoking and non-smoking males and females, reveals a broad spectrum of in vivo somatic mutation occurring in a population of healthy people. Identification of the DNA alteration in individual mutant clones was accomplished using either one or a combination of multiplex polymerase chain reaction analysis of genomic DNA, sequencing of cDNA, and genomic DNA sequencing. The total spectrum consists of 59% (128/217) base substitutions: 126 simple and two tandem CC>TT base substitutions; 39% (85/217) deletion/insertion type mutations: 30 frameshifts, 26 small (3-200 basepairs) and 27 large deletions, and two duplications; and the remaining 2% (4/217) complex mutations involving the deletion of one to 11 basepairs which are replaced by 1 to 10 basepairs. No significant difference was detected between the base substitution spectra for the smokers and the non-smokers. Analysis of the number of mutations occurring at any one base position led to the identification of three hotspots for mutations at basepairs 197, 508 and 617, in the hprt gene coding region. Spontaneous deamination of CpG may be implicated in the creation of basepair 508 as a hotspot since all mutations detected are C>T transitions resulting in the nonsense mutation, TAG. At basepairs 197 and 617 both G>T transversions and G>A transitions were found indicating that at least two mechanisms were involved in creating mutations at these positions. Comparison of the mutation spectra from two populations can provide insight into the origin of the mutations. This study provides an excellent base for comparison of mutation spectra in other human populations.