The carrier protein(s) responsible for mammalian L-system amino acid transport has not been identified. Chronic lymphocytic leukemia (CLL) B-lymphocytes have markedly diminished L-system transport which is restored after treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Six candidate L-system related plasma membrane proteins were identified in TPA-treated CLL-cells using an L-system photoprobe and ultra-high-resolution two-dimensional gel electrophoresis. In the current studies, two candidate L-system related proteins were obtained from preparative giant two dimensional gels, and the amino acid sequences of peptide fragments from these proteins were determined by Edman degradation. These proteins, forming a doublet on the gels at an apparent size of 40 kDa and pI 5.85, had sequence homology to the mitochondrial heat shock protein 60 (hsp60). The presence of these proteins in CLL lymphocyte plasma membranes was confirmed by immunoblotting with antibodies to hsp60. These observations indicate that an hsp60 homologue is associated with the L-system amino acid transporter.