The quinoxaline derivative HBY 097, an orally active nonnucleoside inhibitor of HIV-1 reverse transcriptase (NNRTI), showed an efficient suppression of viral load in a dose-escalating phase I study with mean trough concentrations increasing from 137-1299 ug/l [Rübsamen-Waigmann et al., Lancet 349:1517]. Half-maximal inhibitory concentrations (IC50) for viruses grown from the patients at entry of the study were 0.1-3 nM, except for one patient who had a virus with reduced susceptibility to HBY 097 at entry (IC50: 160 nM). During therapy, only two patients developed a virus with a moderately increased IC50 (2.2 and 15 nM). This reduced susceptibility was associated with the known NNRTI-resistance mutation K ==> N at position 103, in contrast to resistance selection in vitro, which had yielded predominant mutations at positions 179 and 190. The Tyr mutation at position 181, inducing high resistance for other NNRTIs, was never observed. The resistant virus at study entry (IC50 = 160 nM) had a mutation at position 103 as well, combined with an AZT resistance mutation (K ==> R) at position 70, suggesting that nucleoside-resistance mutations may help increasing resistance to HBY 097. This is in line with our in vitro selection studies, where resistance mutations at the 'nucleoside sites' 74 and 75 increased the resistance phenotype of NNRTI mutations. Our findings highlight the crucial importance of IC50 determinations from cultured virus for determination of phenotypic resistance development during therapy and demonstrate that in vivo resistance development cannot be predicted from in vitro selection.