The in vitro activities of amphotericin B (AmB) were evaluated against 40 isolates of Cryptococcus neoformans using time-kill curves. The isolates were obtained from 20 AIDS patients with cryptococcal meningitis submitted to AmB therapy. Isolates were exposed in vitro to 1 microg/mL of AmB that represents a serum concentration of AmB, and the viable colony counts were determined over time. AmB exhibited fungicidal activity at 6 and 12 h for 70.6% of isolates, at 24 h for 7.3%, and at 48 h for 22% of isolates, respectively. This effect was not maximized when the test drug concentration was up to 4 times the AmB MIC for the isolates. Regrowth was observed in 17.5% of the isolates after fungicidal endpoint. With standard in vitro susceptibility testing, this tolerance phenomenon could not be assessed, and thus, these tests may underestimate the resistance of C. neoformans to AmB in vivo. AmB is the first-choice drug for the treatment of cryptococcosis in Brazil, and future studies using time-kill methodology are needed to estimate the predictive value of this test in the clinical failure.