Since the 1990s, the substantial increase in the rate of Candida glabrata infections has become a serious problem. As most C. glabrata infections arise from the host's endogenous microflora, the present prospective, multicenter analysis included all clinical isolates associated with colonization and with systemic and hematogenous candidiasis. Among 347 C. glabrata isolates, the overall rates of resistance to fluconazole (MIC ≥ 64 μg/ ml) and itraconazole (MIC ≥ 1 μg/ml) were 10.7 and 15.2%, respectively, although for half (n = 148) of the itraconazole-susceptible isolates the MICs (0.25 to 0.5 μg/ml) were in the susceptible-dependent upon dose range. Fluconazole resistance was more common among C. glabrata isolates obtained from centers caring for patients with cancer (MICs at which 90% of isolates are inhibited [MIC90s] = 32 μg/ml) or AIDS (MIC90s > 64 μg/ml) than among C. glabrata isolates from a community-based university medical center (MIC90s = 16 μg/ ml) (P = 0.001). Thirty-three bloodstream isolates and those obtained from other body sites had similar in vitro susceptibility profiles. The fluconazole MIC90s (≤16 μg/ml) for C. glabrata yeast isolates from the gastrointestinal tract were lower than those (≥64 μg/ml) for C. glabrata isolates from respiratory and urinary tract samples (P = 0.01). A similar discrepancy for itraconazole was not significant (P > 0.5). We did not observe differences in fluconazole or itraconazole susceptibility profiles among C. glabrata isolates associated with either hematogenous dissemination or colonization. The significant discrepancy in antifungal susceptibility among C. glabrata organisms isolated from hospitals in the same geographic region emphasizes the significance of periodic susceptibility surveillance programs for individual institutions, especially those providing care to patients at risk.