Whether or not intracellular transport and surface expression of the invariant chain (Ii; CD74) occurs independent of the presence of major histocompatibility complex (MHC) class II molecules was examined by comparing the class II-negative mutant lymphoblastoid cell line 174 x CEM.T2 (T2) and its class II-positive parental cell line 174 x CEM.T1 (T1). We found a similar proportion of Ii being transported to the Golgi complex in T1 and T2, as monitored by the degree of sialic acid addition to glycan side chains of Ii. In agreement with this result, T1 and T2 expressed comparable amounts of Ii at the cell surface, as measured by flow cytometry. This indicates that, although not associated with class II molecules, a proportion of Ii is transported to the plasma membrane. Both in T1 and T2, surface Ii (sIi) was rapidly internalized with a half-life of 3-4 min, suggesting that some Ii enters the endocytic route via the cell surface after being internalized. Our data demonstrate transport of Ii on a route alternative to the endocytic pathway. This alternative route could also account for delivery of newly synthesized class II-Ii complexes to processing compartments in antigen-presenting cells.