The fungal opportunist Cryptococcus neoformans forms biofilms in vitro and in vivo. C. neoformans has an unusual ability to grow over a wide range of temperatures, and is one of only two species in the genus able to grow at 37 degrees C. The optimum growth temperature in the laboratory is 30 degrees C, but Clinical and Laboratory Standards Institute (CLSI) planktonic susceptibility testing is performed at 35 degrees C. We investigated whether these growth temperatures affected C. neoformans biofilm formation and drug resistance. Biofilms of 30 strains of C. neoformans were grown at 30 degrees C or 35 degrees C, and antifungal susceptibilities evaluated at 30 degrees C or 35 degrees C using minimum biofilm eradication endpoints. At 35 degrees C, biofilms from 40% of the strains were more susceptible to flucytosine, 30% were more susceptible to nystatin, 27% were more susceptible to amphotericin, and 20% were more susceptible to fluconazole, as compared to 30 degrees C. The reverse, that is an increased susceptibility at 30 degrees C, only occurred with a single strain using nystatin or fluconazole. For the remaining strains, biofilm susceptibility was equivalent at the two temperatures. Biofilm colony forming units (CFU)s, as measured indirectly by 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction, were greater at 35 degrees C than at 30 degrees C for the majority of the strains. Thus, growth temperature does affect C. neoformans biofilm properties, but factors other than relative biofilm CFUs/ml must be involved in the increased drug susceptibility at 35 degrees C.