Within-line heterogeneity has been found in the mitochondrial DNA (mtDNA) in two isofemale lines of D. simulans. The co-existing types, S and M, were typical of the mtDNA in D. simulans and in D. mauritiana, respectively, their nucleotide divergence per site being ca. 2·1%. Segregation analysis confirmed that some individuals in these lines were heteroplasmic and suggested incomplete maternal inheritance of mtDNA in Drosophila. Examination of other lines of D. simulans revealed that the M type of D. mauritiana occurs at 71% in Réunion, 38% in Madagascar and 0% in Kenya. This finding and interspecific sequence comparisons of both M types indicate that D. mauritiana diverged from D. simulans probably less than 240000 years ago.