Fungi belonging to the recently classified genus Pseudozyma possess some unique properties such as biocontrol activity, production of rare antimicrobial glycolipids and production of recombinant proteins. In this work, we report the first cloning of a promoter endogenous to the multi-faceted yeast-like Pseudozyma flocculosa, that of the actin gene. The promoter region lacked typical TATA or CAAT box but displayed three putative GC box and two CT-rich regions. As in other related basidiomycetes, only one copy of the actin gene was present in the genome of P flocculosa. The activity of the actin promoter was compared to that of the HSP70 promoter from Ustilago maydis in two Pseudozyma species. In R flocculosa, the actin promoter allowed the expression of a very high amount of GFP protein (27.8 mg g(-1) total protein) compared to those obtained with the HSP70 promoter in liquid culture. By contrast, the levels of GFP expression obtained in liquid culture were similar with the actin or the HSP70 promoter in Pseudozyma antarctica. A similar pattern of GFP expression was observed in solid culture. The cloning of this new promoter offers a unique genetic tool to further exploit and study the unusual properties of fungi from the Pseudozyma genus.