The membrane topology of two alkane-inducible cytochromes P450 from the yeast Candida tropicalis, alk1 and alk2, was tested by construction of fusion proteins with part of invertase and histidinol dehydrogenase (invHIS4C) and expression in a Saccharomyces cerevisiae his4 mutant. Depending on the localization of invHIS4C on the endoplasmic reticulum (ER) cytoplasmic or luminal side, the enzyme converts histidinol to histidine and allows the his4 yeast strain to grow on histidinol-supplemented medium. The N-terminal segments of alk1 and alk2 were fused to invHIS4C at three different locations that follow the first alk1 and alk2 transmembrane domains or a second putative transmembrane domain of alk1. The combination of this in vivo assay with subcellular immunoprecipitations of the expressed fusion proteins allowed us to establish that both P450s contain only one transmembrane domain with their N-terminus located in the ER lumen. Deletions performed in these fusion proteins removing the first transmembrane domain of alk1 (TM) resulted in a less efficient targeting to the ER membrane but did not prevent their insertion in these membranes. Furthermore deletion of a negatively charged peptide preceding the first alk1 transmembrane domain (L) in an invHIS4C protein fused after this domain caused the N-terminal to have a positive net charge and to be oriented in the cytoplasm thus translocating the remaining protein into the ER lumen. The presence of the second hydrophobic segment, however, prevented the complete translocation of this fusion protein into the ER lumen. This study describes the first assessment of P450 membrane topology using an in vivo technique.