Apolipoprotein M (apoM) in human plasma is mainly associated with HDL. A retained signal peptide anchors apoM to the lipoproteins. To investigate the role of the signal peptide in the transfer of apoM from the synthesizing cell to the lipoproteins, wildtype apoM cDNA and the Q22A mutant, introducing a signal peptidase cleavage site, were used to stably transfect HEK293 cells, which intrinsically do not express apolipoproteins. When cultured under serum-free conditions, wildtype apoM was, in contrast to Q22A, poorly secreted. Addition of serum or purified HDL stimulated secretion of wildtype apoM, which was recovered in the medium incorporated in HDL. The liver cell line HepG2, which synthesizes HDL, was cultured under serum-free conditions and found to secrete apoM as part of an HDL-like particle. In conclusion, due to its retained signal peptide, apoM is poorly secreted unless HDL is either coexpressed or added to the culture medium.