The methylotrophic yeasts have been the subject of intensive studies, because of their highly regulated methanol metabolism and the biogenesis of peroxisomes. We investigated the 5' regulatory region of the MOX gene from the yeast, Hansenula polymorpha, encoding the peroxisomal methanol oxidase, the key enzyme of methanol metabolism. This tightly regulated yeast promoter of approximately 1.5 kb is unusually large, and also of remarkable strength under inducing conditions, belonging to the strongest yeast promoters yet described. Deletion analyses revealed a complex promoter structure composed of several sequence elements with positive and negative regulatory effects on reporter gene expression and a pronounced cooperation between the elements. Specific binding of several factors was detected in vitro by gel retardation and DNase I footprinting experiments. On the basis of deletion data, two binding sites could be identified as upstream activation sequences (UAS1 and UAS2) and one binding site as an upstream repressing sequence (URS1).