Our understanding of lysosomal biogenesis and general vesicular transport in animal cells has been greatly enhanced by studies of vacuolar biogenesis in yeast. Genetic screens have identified a number of proteins that play direct roles in the proper sorting of vacuolar hydrolases. These include t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and Sec1p-like proteins, which have recently been implicated as key regulators of vesicle fusion. In this study we have extended these observations in yeast and have isolated and characterized a novel member of the Sec1p-like family of proteins from mammalian cells, mVps45. mVps45 shares a high level of identity with the Saccharomyces cerevisiae Sec1p-like protein Vps45p that is believed to function with the t-SNARE Pep12p in the fusion of Golgi-derived transport vesicles with a prevacuolar compartment. We found that mVps45 is a ubiquitously expressed peripheral membrane protein that localized to perinuclear Golgi-like and trans-Golgi network compartments in Chinese hamster ovary cells. We found that mVps45 could bind specifically to yeast Pep12p and to the mammalian Pep12p-like protein, syntaxin 6, in vitro.