CD81 is a widely expressed tetraspanin that associates in B cells with CD19 in the CD19-CD21-CD81 signaling complex. CD81 is necessary for normal CD19 expression; cd81−/− B cells express lower levels of CD19, especially cd81−/− small pre-BII cells, which are almost devoid of surface CD19. The dependence of CD19 expression on CD81 is specific to this particular tetraspanin since cd9−/− B cells express normal levels of CD19. Furthermore, expression of human CD81 in mouse cd81−/− B cells restored surface CD19 to normal levels. Quantitative analysis of CD19 mRNA demonstrated normal levels, even in cd81−/− pre-BII cells. Analysis of CD19 at the protein level identified two CD19 glycoforms in both wild-type and cd81−/− B cells. The higher M r glycoform is significantly reduced in cd81−/− B cells and is endoglycosidase H (endo-H) resistant. In contrast, the low M r glycoform is comparably expressed in cd81−/− and in wild-type B cells and is endo-H sensitive. Because endo-H sensitivity is tightly correlated with endoplasmic reticulum localization, we suggest that the dependency of CD19 expression on CD81 occurs in a postendoplasmic reticulum compartment where CD81 is necessary for normal trafficking or for surface membrane stability of CD19.