The endoplasmic reticulum membrane protein SREBP cleavage-activating protein (Scap) senses sterols and regulates activation of sterol-regulatory element binding proteins (SREBPs), membrane-bound transcription factors that control lipid homeostasis in fission yeast and mammals. Transmembrane segments 2-6 of Scap function as a sterol-sensing domain (SSD) that recognizes changes in cellular sterols and facilitates activation of SREBP. Previous studies identified conserved mutations Y298C, L315F, and D443N in the SSD of mammalian Scap and fission yeast Scap (Scp1) that render cells insensitive to sterols and cause constitutive SREBP activation. In this study, we utilized fission yeast genetics to identify additional functionally important residues in the SSD of Scp1 and Scap. Using a site-directed mutagenesis selection, we sampled all possible amino acid substitutions at 50 conserved residues in the SSD of Scp1 for their effects on yeast SREBP (Sre1) activation. We found mutations at 23 different amino acids in Scp1 that rendered Scp1 insensitive to sterols and caused constitutive activation of Sre1. To our surprise, the majority of the homologous Scap mutants displayed wild-type function, and only one mutation, V439G, caused constitutive activation of SREBP in mammals. These results suggest that the sterol-sensing mechanism of Scap and the functional requirements for SREBP activation are different between fission yeast and mammals.