The tyrosinase family of genes has been conserved throughout vertebrate evolution. The role of conserved N-glycan sites in sorting, stability, and activity of tyrosinase family proteins was investigated using two family members from two different species, mouse gp75/tyrosinase-related protein (TRP)-1/Tyrp1 and human tyrosinase. Potential N-linked glycosylation sites on the lumenal domains of mouse gp75/TRP-1/Tyrp1 and human tyrosinase were eliminated by site-directed mutagenesis (Asn to Gln substitutions). Our results show that selected conserved N-glycan sites on tyrosinase family members are crucial for stability in the secretory pathway and endocytic compartment and for enzymatic activity. Different glycan sites on the same tyrosinase family polypeptide can perform distinct functions, and conserved sites on tyrosinase family paralogues can perform different functions.