Protein 4.1B is a membrane skeletal protein expressed in various organs, and is associated with tumor suppressor in lung cancer-1 (TSLC1) in vitro. Although involvement of 4.1B in the intercellular junctions and tumor-suppression was suggested, some controversial results posed questions to the general tumor-suppressive function of 4.1B and its relation to TSLC1 in vivo. In this study, the expression of 4.1B and its interaction with TSLC1 were examined in rodent adrenal gland, and the involvement of 4.1B in tumorigenesis and the effect of 4.1B deficiency on TSLC1 distribution were also investigated using rodent pheochromocytoma and 4.1B-knockout mice. Although plasmalemmal immunolocalization of 4.1B was shown in chromaffin cells of rodent adrenal medulla, expression of 4.1B was maintained in developed pheochromocytoma, and morphological abnormality or pheochromocytoma generation could not be found in 4.1B-deficient mice. Furthermore, molecular interaction and colocalization of 4.1B and TSLC1 were observed in mouse adrenal gland, but the immunolocalization of TSLC1 along chromaffin cell membranes was not affected in the 4.1B-deficient mice. These results suggest that the function of 4.1B as tumor suppressor might significantly differ among organs and species, and that plasmalemmal retention of TSLC1 would be maintained by molecules other than 4.1B interacting in rodent chromaffin cells.