ApoM is a novel apolipoprotein that is reportedly necessary for pre-beta HDL formation; however, its detailed function remains unknown. We investigated the biogenesis and properties of apoM and its effects on the initial steps of nascent pre-beta HDL assembly by ABCA1 in HEK293 cells. Transiently transfected apoM was localized primarily in the endomembrane compartment. Pulse-chase analyses demonstrated that apoM is inefficiently secreted, relative to human serum albumin, and that ~50% remains membrane-associated after extraction with sodium carbonate, pH 11.5. To investigate the role of apoM in nascent pre-beta HDL formation, ABCA1-expressing or control cells, transfected with empty vector, apoM or carboxyl terminal epitope-tagged apoM (apoM-C-FLAG), were incubated with 125I-apoA-I for 24 hours. Conditioned media were harvested and fractionated by FPLC to monitor HDL particle size. Pre-beta HDL particles were formed effectively in the absence of apoM expression; however, increased apoM expression stimulated the formation of larger-sized nascent pre-beta HDLs. Immunoprecipitation with anti-apoA-I antibody followed by apoM Western blot analysis revealed that little secreted apoM was physically associated with pre-beta HDL. Our results suggest that apoM is an atypical secretory protein that is not necessary for ABCA1-dependent pre-beta HDL formation but does stimulate the formation of larger sized pre-beta HDL. We propose that apoM may function catalytically at an intracellular site to transfer lipid onto pre-beta HDL during or after their formation by ABCA1.