Thirteen isolates of human immunodeficiency virus type 1 (HIV-1) obtained in coculture with peripheral blood lymphocytes were tested for in vitro susceptibility to zidovudine (ZDV). Seven isolates were obtained from patients who had never been treated with ZDV and six from patients receiving the drug. The seven isolates from untreated patients and four of six from treated patients were susceptible to ZDV. The two isolates from the patients treated for the longest periods were resistant to the drug. The presence of mutations at critical positions of the reverse transcriptase gene was investigated by direct sequencing of polymerase chain reaction (PCR)-amplified DNA and four isolates were found to be mutants. An isolate from an untreated patient showed a change at residue 70 of the reverse transcriptase and an isolate from a patient treated for 4 months showed a change at residue 67. A change at residue 215 was found only for the two drug-resistant isolates, which correlated with the results obtained by Larder et al. using isolates from MT-2 cell cocultures. These results suggest that any HIV isolate provided by conventional coculture could be confidently tested for ZDV susceptibility in order to study the emergence of resistance during long-term therapy.