Fps1p is a glycerol efflux channel from Saccharomyces cerevisiae. In this atypical Major Intrinsic Protein (MIP) neither of the family's signature NPA motifs, which are part of the pore, is preserved. In order to understand the functional consequences of this feature, we analyzed the pseudo-NPA motifs of Fps1p by site-directed mutagenesis and assayed the resultant mutant proteins in vivo. In addition, we took advantage of the fact that Fps1p's closest bacterial homolog, E. coli GlpF, can be functionally expressed in yeast, thus enabling the analysis in yeast cells of mutations that make this typical MIP more similar to Fps1p. We observed that mutations made in Fps1p to 'restore' the signature NPA motifs did not substantially affect channel function. In contrast, when GlpF was mutated to resemble Fps1p all mutants had reduced activity compared to wild-type. We rationalized these data by constructing models of one GlpF mutant and of the transmembrane core of Fps1p. Our model predicts that the pore of Fps1p is more flexible than that of GlpF. We discuss the fact that this may accommodate Fps1p's divergent NPA motifs, and that the different pore structures of Fps1p and GlpF may reflect the physiological roles of the two glycerol facilitators.