The evolutionary rate of the human T-cell lymphotropic virus type-1 (HTLV-1) is considered to be very low, in strong contrast to the related human retrovirus HIV. However, current estimates of the HTLV-1 rate rely on the anthropological calibration of phylogenies using assumed dates of human migration events. To obtain an independent rate estimate, we analyzed two variable regions of the HTLV-1 genome (LTR and env) from eight infected families. Remarkable genetic stability was observed, as only two mutations in LTR (756 bp) and three mutations in env (522 bp) occurred within the 16 vertical transmission chains, including one ambiguous position in each region. The evolutionary rate in HTLV-1 was then calculated using a maximum-likelihood approach that used the highest and lowest possible times of HTLV-1 shared ancestry, given the known transmission histories. The rates for the LTR and env regions were 9.58 x 10(-8)-1.25 x 10(-5) and 7.84 x 10(-7) -2.33 x 10(-5)nucleotide substitutions per site per year, respectively. A more precise estimate was obtained for the combined LTR-env data set, which was 7.06 x 10(-7)-1.38 x 10(-5)substitutions per site per year. We also note an interesting correlation between the occurrence of mutations in HTLV-1 and the age of the individual infected.