Development of stavudine resistance was studied using human immunodeficiency virus type 1 isolates from 13 patients treated with stavudine for 18-22 months. Drug sensitivity testing on 11 of these pre- and posttherapy isolates identified only 2 posttreatment isolates with decreased stavudine sensitivity (ED50s < 4-fold higher than the average pretreatment ED50). Genotypic analysis of all 13 pairs of isolates identified multiple mutations in the reverse transcriptase (RT) gene. However, no genetic basis was identified to account for the observed changes in stavudine susceptibility. A recombinant virus containing the entire RT gene of the posttherapy isolate displaying the greatest resistance remained sensitive to stavudine. Five of the stavudine posttreatment isolates developed resistance (9- to 176-fold) to zidovudine, although the relationship between stavudine treatment and the appearance of zidovudine resistance remains unexplained. Analysis of 10 additional pairs of isolates did not confirm this relationship. The low frequency and modest degree of change in stavudine sensitivity following prolonged treatment is very encouraging.