Band 4.2 (pallidin) is a major erythrocyte membrane protein which has been detected in a number of nonerythroid cell types. Increasing evidence suggests that band 4.2 is involved in maintaining membrane stability in the erythrocyte. For example, band 4.2 binds to the integral membrane protein band 3 and to cytoskeletal proteins in the erythrocyte membrane, and band 4.2 deficiency results in varying degrees of hemolytic anemia. We have previously shown that human erythrocyte band 4.2 is myristylated at its penultimate glycine. Here we report that when expressed in both Sf9 and COS cells, myristylated forms of band 4.2 are detected at different intracellular locations than nonmyristylated forms. We also show that the unspliced form of human erythrocyte band 4.2 (a minor form in reticulocytes which contains an additional 30 amino acids after the first three N-terminal amino acids compared to the major erythroid form) is myristylated only at a barely detectable level, while mouse erythrocyte band 4.2 (homologous to the major erythroid form of human band 4.2) is myristylated at a level comparable to that of human band 4.2. These results suggest that myristylation plays a key role in the targeting of band 4.2 to specific intracellular locations and is likely to have a role in the function of this protein.