Mark Bretscher initially studied protein synthesis, discovering the intermediate peptidyl-tRNA. He also helped to understand chain initiation and termination and proposed a hybrid site structure for ribosomes during translocation.

His most important work concerns biological membranes. Using novel chemical methods on human erythrocytes, he was the first to demonstrate the existence of oriented transmembrane proteins. He also discovered that the bilayer structure is asymmetric with amino phospholipids restricted to the inner leaflet. This research led him to propose new principles of biological membrane structure, which are widely accepted. He also suggested that the length of the transmembrane domain of a membrane protein determines whether it resides in the Golgi apparatus or plasma membrane.

Mark discovered that, in motile cells, membrane endocytosed by coated pits is returned to the cell surface at the cell’s leading edge. However, if sessile, it is returned randomly to the cell’s surface. This provides a basis for his proposals of how animal cells migrate and, when they do so, are able to replenish a source of adhesion sites at their fronts.