Five histone deacetylase genes (HDA1, RPD3, HOS1, HOS2, and HOS3) have been cloned from Candida albicans and characterized. Sequence analysis and comparison with 17 additional deacetylases resulted in a phylogenetic tree composed of three major groups. Transcription of the deacetylases HDA1 and RPD3 is down-regulated in the opaque phase of the white-opaque transition in strain WO-1. HOS3 is selectively transcribed as a 2.5-kb transcript in the white phase and as a less-abundant 2.3-kb transcript in the opaque phase. HDAI and RPD3 were independently deleted in strain WO-1, and both switching between the white and opaque phases and the downstream regulation of phase-specific genes were analyzed. Deletion of HDA1 resulted in an increase in the frequency of switching from the white phase to the opaque phase, but had no effect on the frequency of switching from the opaque phase to the white phase. Deletion of RPD3 resulted in an increase in the frequency of switching in both directions. Deletion of HDA1 resulted in reduced white-phase-specific expression of the EFG1 3.2-kb transcript, but had no significant effect on white-phase-specific expression of WH11 or opaque-phase-specific expression of OP4, SAP1, and SAP3. Deletion of RPD3 resulted in reduced opaque-phase-specific expression of OP4, SAP1, and SAP3 and a slight reduction of white-phase-specific expression of WH11 and 3.2-kb EFG1. Deletion of neither HDA1 nor RPD3 affected the high level of white-phase expression and the low level of opaque-phase expression of the MADS box protein gene MCM1, which has been implicated in the regulation of opaque-phase-specific gene expression. In addition, there was no effect on the phase-regulated levels of expression of the other deacetylase genes. These results demonstrate that the two deacetylase genes HDA1 and RPD3 play distinct roles in the suppression of switching, that the two play distinct and selective roles in the regulation of phase-specific genes, and that the deacetylases are in turn regulated by switching.