Candida albicans, like other pathogens, can form complex biofilms on a variety of substrates. However, as the number of studies of gene regulation, architecture, and pathogenic traits of C. albicans biofilms has increased, so have differences in results. This suggests that depending upon the conditions employed, biofilms may vary widely, thus hampering attempts at a uniform description. Gene expression studies suggest that this may be the case. To explore this hypothesis further, we compared the architectures and traits of biofilms formed in RPMI 1640 and Spider media at 37°C in air. Biofilms formed by a/⣠cells in the two media differed to various degrees in cellular architecture, matrix deposition, penetrability by leukocytes, fluconazole susceptibility, and the facilitation of mating. Similar comparisons of a/a cells in the two media, however, were made difficult given that in air, although a/a cells form traditional biofilms in RPMI medium, they form polylayers composed primarily of yeast cells in Spider medium. These polylayers lack an upper hyphal/matrix region, are readily penetrated by leukocytes, are highly fluconazole susceptible, and do not facilitate mating. If, however, air is replaced with 20% CO 2 , a/a cells make a biofilm in Spider medium similar architecturally to that of a/⣠cells, which facilitates mating. A second, more cursory comparison is made between the disparate cellular architectures of a/a biofilms formed in air in RPMI and Lee's media. The results demonstrate that C. albicans forms very different types of biofilms depending upon the composition of the medium, level of CO 2 in the atmosphere, and configuration of the MTL locus.T he opportunistic fungal pathogen Candida albicans, like opportunistic bacterial pathogens (1, 2), forms biofilms on the surfaces of a variety of tissues and synthetic devices introduced into hosts (3-5). Traditionally, biofilms serve the purpose of anchoring populations of pathogens to a surface and providing a conditioned microenvironment that facilitates growth, survival, and intercellular signaling (6, 7). Biofilms also serve to exclude host and therapeutic challenges, such as antibodies, white blood cells, and antibiotics (8, 9). Bacterial biofilms, which were initially the most intensely studied, have been shown to be architecturally complex, adhering at their base to a targeted surface and composed of multiple cell types embedded in a self-generated complex extracellular matrix (10, 11). Many of the characteristics of bacterial biofilms have been shown to be exhibited by biofilms formed by C. albicans, including drug resistance (12-15), impermeability to low-and high-molecular-weight molecules (15), and resistance to penetration by human polymorphonuclear leukocytes (PMNs) (15, 16). However, studies both to characterize the traits of C. albicans biofilms and to elucidate the molecular mechanisms regulating formation do not always provide the same results, most notably transcription profiles, mutational analyses of the signal transduction pathways, and t...