Lysosomal enzymes containing mannose 6‐phosphate recognition markers are sorted to lysosomes by mannose 6‐phosphate receptors (MPRs). The physiological importance of this targeting mechanism is illustrated by I‐cell disease, a fatal lysosomal storage disorder caused by the absence of mannose 6‐phosphate residues in lysosomal enzymes. Most mammalian cells express two MPRs. Although the binding specificities, subcellular distribution and expression pattern of the two receptors can be differentiated, their coexpression is not understood. The larger of the two receptors with an M(r) of approximately 300,000 (MPR300), which also binds IGFII, appears to have a dominant role in lysosomal enzyme targeting, while the function of the smaller receptor with an M(r) of 46,000 (MPR46) is less clear. To investigate the in vivo function of the MPR46, we generated MPR46‐deficient mice using gene targeting in embryonic stem cells. Reduced intracellular retention of newly synthesized lysosomal proteins in cells from MPR46 ‐/‐ mice demonstrated an essential sorting function of MPR46. The phenotype of MPR46 ‐/‐ mice was normal, indicating mechanisms that compensate the MPR46 deficiency in vivo.