The objective of this study was to evaluate the activity of efavirenz after the failure of a nevirapine-containing regimen. This prospective study included 47 patients with plasma HIV loads >1000 copies/ml, and who had received nevirapine for at least 16 weeks, included in an efavirenz-based salvage regimen. The main outcome measure was virological response, defined as an HIV RNA level decrease of at least 1 log(10) copies/ml after 24 weeks, according to genotypic and phenotypic resistance to efavirenz. Phenotypic resistance was defined as a >10-fold increase in the IC(50). The median CD4(+) cell count was 236 x 10(6)/liter and the median HIV RNA level was 4.5 log(10) copies/ml. Mutations known to decrease susceptibility to nonnucleoside reverse transcriptase inhibitors were observed in 79% of patients, predominantly at residues 181 (49%), 103 (40%), and 106 (19%), but phenotypic resistance to efavirenz was seen in 62% of cases. All the strains with the K103N mutation showed high-level resistance to efavirenz, in contrast with 20% of those carrying exclusively the Y181C mutation. By week 24, 38% of patients had responded and 19% had achieved an undetectable HIV load. Virological failure was observed in patients with phenotypic resistance to efavirenz (67 vs. 11%; relative risk [RR], 4; 95% confidence interval [CI], 1.07-14.89; p = 0.04), or in presence of the K103N mutation (52 vs. 17%; RR, 1.77; 95% CI, 1.12-2.79; p = 0.02), and these results remained unchanged after adjusting for HIV load, or by resistance to the accompanying drugs in the salvage regimen. A previous longer period of nevirapine therapy was significantly associated with the emergence of efavirenz resistance (288 vs. 170 days, p < 0.01). We conclude that genotypic and/or phenotypic resistance assays permit the sequential use of nonnucleoside reverse transcriptase inhibitors in the clinical setting. Our data suggest that an early change after nevirapine failure could avoid the emergence of efavirenz resistance.