The protein RHAMM has roles in cell signaling, migration and adhesion and mediates processes, in part, via interactions with hyaluronan (HA), microtubules, actin, calmodulin and components of the ERK signaling pathway. RHAMM appears to be regulated by alternate splicing, which may be responsible for targeting of RHAMM to intracellular and plasma membrane sites. A role for RHAMM in neural cells is supported by reports of modified neurite extension and glial cell migration in response to anti-RHAMM antibodies and peptides corresponding to sequence in RHAMM. This study describes the initial characterization of RHAMM in the CNS. Northern blotting of rat brain and PC12 cell RNA revealed an apparent single band which concurred with the single RHAMM sequence identified by RT-PCR and was homologous to human RHAMM. No evidence of alternate splicing was found. For analysis of RHAMM at the protein level, antibodies were generated against sequence in RHAMM. Western blots of brain and PC12 cell homo enates revealed several RHAMM isoforms. Immunohistochemically, RHAMM was present in neurons and oligodendrocytes of adult brain, where heterogenous RHAMM expression was common in cell populations, suggesting regulated expression. Subcellular fractionation of brain indicated RHAMM is localized intracellularly. The novel finding of a RHAMM form in brain mitochondria was confirmed by confocal microscopy. RHAMM was demonstrated to interact with ERK and calmodulin. These findings, together with observations of RHAMM association with microfilaments and microtubules suggest that RHAMM may act as a signaling scaffold for targeting of signaling molecules to mitochondria and the cytoskeleton. Further, our results suggest as yet unidentified intracellular functions of HA.