Rhomboid peptidases are conserved intramembrane serine proteases with mitochondrial family members being involved in mitochondrial dynamics and apoptosis. The Saccharomyces cerevisiae mitochondrial rhomboid, Pcp1p, catalyzes the cleavage of two substrates: Ccp1p, which breaks down reactive oxygen species, and Mgm1p, a GTPase mediating mitochondrial fusion events. As an initial approach to determine the structural basis of Pcp1p activity, a screen to identify temperature sensitive alleles of PCP1 was performed using hydroxylamine mutagenesis. Eight mutants were identified from a pool of 30,000 colonies that exhibited either temperature sensitive growth or respiratory defects. These mutants also exhibited defects in Mgm1p and Ccp1p processing and some degree of abnormal mitochondrial morphology. The majority of amino acid changes occurred within the fourth and sixth transmembrane domains of Pcp1p, the location of the active site serine and histidine residues, supporting a role for these transmembrane helices in Pcp1p activity.