Thirty HIV isolates, obtained from 15 patients before and after receiving single drug therapy with didanosine (ddI), were examined for sensitivity to ddI and zidovudine (ZDV) using a peripheral blood mononuclear leukocyte (PBML)-based assay. Fourteen of the patients had ARC, one had AIDS and 12 had received previous therapy with ZDV. After a median of 1 year of ddI therapy, isolates were significantly less sensitive to ddI than were isolates obtained prior to therapy (P = 0.03). A decrease in ddI sensitivity was observed in ten of the 15 isolate pairs. In contrast to ddI susceptibilities, sensitivity to ZDV increased over the same period of time (P = 0.03). Additional isolates were obtained from four patients who received ddI monotherapy for 2 years. Three of these isolates demonstrated no change in ddI sensitivity compared to baseline. No correlation could be made in this study between development of decreased ddI sensitivity and serum p24 levels, CD4 counts, or clinical outcome. Decreased ddI sensitivity occurs frequently among HIV isolates obtained from long-term recipients of ddI. This decreased sensitivity is modest in degree and is of unknown clinical significance.