EFG1, which encodes a trans-acting factor, is expressed as a more abundant 3.2 kb transcript in the white phase and as a less abundant 2.2 kb transcript in the opaque phase of the white-opaque transition in Candida albicans. To understand how alternative phase-specific mRNAs are transcribed from the same gene locus, the 2320 bp upstream region of the gene was functionally characterized by analysing the -activity of deletion derivatives in a luciferase-based reporter system. The white phase-specific promoter contained three discrete sequences involved in white phase-specific activation, between -2022 and -1809 bp (AR1), between -1809 and -1727 bp (AR2) and between -922 and -840 bp (AR3). A higher resolution deletion and mutation analysis of AR2 revealed two regions between -1809 and -1787 bp and between -1764 and -1728 bp that are responsible for AR2 activation. Targeting of promoter constructs to the ectopic ADE2 genomic site and the 3' end of the EFG1 genomic site revealed a positional requirement for white phase-regulated activation specific for the AR2 region of the promoter. Gel mobility shift assays using AR2 revealed a white phase-specific activation complex. No discrete activation sequences were identified in the overlapping promoter of the opaque phase-specific EFG1 transcript. The strength of opaque phase activation was directly proportional to the length of the promoter. Northern analysis excluded the possibility of an opaque phase-specific repressor. These results demonstrate overlapping promoters for white and opaque phase-specific expression of the gene for the transcription factor Efg1, with distinctly different mechanisms of phase-specific activation.