Emergence of resistance to antifungal drugs during therapy for invasive aspergillosis has received scant attention. We recovered Aspergillus isolates from six patients with invasive aspergillosis, who were receiving amphotericin B before fungal isolation. Although isolates were susceptible to amphotericin B in vitro, none of the patients survived. The MIC of amphotericin B for isolates was similar to that for isolates from 35 patients with no prior exposure to amphotericin B. Laboratory attempts to produce amphotericin B resistance in Aspergillus were unsuccessful. These data indicate that emergence of resistance to amphotericin B is uncommon during therapy for invasive aspergillosis.