Cross-resistance to nelfinavir (NFV) is observed in patients failing protease inhibitor (PI)-containing therapies. We performed a study with 111 patients who started an NFV-based salvage regimen after failing PI-based therapy to evaluate genotypic changes and to identify factors associated with resistance to NFV. Genotypic and phenotypic resistance data at entry (111 and 51 samples) and after NFV failure (74 and 31 samples) were available. Median CD4(+) cell count was 208 x 10(6)/liter, HIV RNA level was 4.6 log(10) copies/ml, and median number of mutations in the protease was 9. At baseline, 51 and 14% of viral isolates showed high or intermediate phenotypic resistance to NFV. Phenotypic data correlated with virological outcome, reaching undetectability at the third month in 40, 14, and 0% of those patients with susceptible, intermediate, or resistant viral isolates, respectively. Phenotypic resistance to NFV was associated with the presence of the L90M mutation: 46% for resistant vs. 6% in susceptible strains. The number of mutations in the protease correlated with the fold-increase in the IC(50)-NFV. The D30N mutation was detected in only 1 of 74 patients who failed. In a logistic regression analysis, the number of mutations in the protease was associated with NFV cross-resistance (RR, 2.09 per each additional mutation; 95% CI 1.23-3.55; p < 0.01). In conclusion, phenotypic cross-resistance to NFV for PI-experienced patients can be predicted by the number of mutations in the protease. The L90M mutation is significantly associated with the subsequent failure of NFV-containing regimens. The presence of the D30N mutation was rare and not useful in identifying NFV-resistant isolates.