The metalloproteome is defined as the set of proteins which have metal-binding capacity, by being metalloproteins or having metal-binding sites. A different metalloproteome may exist for each metal. Mass spectrometric characterization of metalloproteomes provides valuable information relating to cellular disposition of metals physiologically and in metal-associated diseases. We examined the Cu- and Zn-metalloproteomes in three human hepatoma lines: Hep G2 and Mz-Hep-1, which retain many functional characteristics of normal human hepatocytes, and SK-Hep-1, which is poorly differentiated. Additionally we studied a single specimen of normal human liver and Hep G2 cells depleted in vitro of cellular copper. We used MALDI and ESI QTOF mass spectrometry to analyze peptide sequences of tryptic digests obtained by either in-gel digestion of metal-binding proteins or peptides on an IMAC column loaded with either Cu or Zn. Mainly high abundance proteins were identified. Cu-binding proteins identified included enolase, albumin, transferrin and alcohol dehydrogenase and certain intracellular chaperone proteins. The Cu-metalloproteome was not identical to the Zn-metalloproteome. Peptide binding experiments demonstrated that Cu-coordination prefers the order of residues: histidine > methionine > cysteine Although the Cu-metalloproteome was similar from line to line, subtle differences were apparent. Gel profiling showed more extensive variation in expression of annexin II in SK-Hep-1 and Mz-Hep-1 than in Hep G2 and normal liver tissue. Glycerylphosphoryl- ethanolamine was identified as a post-translational modification at residue Glu 301 of elongation factor 1-a in Hep G2. Intracellular copper depletion was associated with loss of the glycerylphosphoryl side group. These findings suggest that post-translational modification could be affected by intracellular actions of copper. Comparison of the Cu- and Zn-metalloproteomes in Hep G2 with a published general proteome of Hep G2 disclosed little overlap. Proteins in the metalloproteomes of human hepatocytes can be identified by these methods. Variations in these metalloproteomes may have important physiological relevance.