In order to investigate heterosis on a molecular basis, suppression subtractive hybridization was used to analyze the differences in gene expression between porcine F1 hybrids Landrace x Yorkshire and their female parents Yorkshire. From two specific subtractive cDNA libraries, the clones screened out by reverse Northern high-density blots screening were chosen to clone full-length cDNA by RACE. An expression-upregulated gene for Yorkshire skeletal muscle, designated as HUMMLC2B, was identified. Porcine HUMMLC2B contains an open reading frame (ORF) encoding 169 amino acids residues with 59 and 115 nucleotides in the 5' and 3' untranslated regions (UTRs), respectively. In the porcine genome, it contains seven exons separated by six introns. High allelic variations and four SINEs were detected in it. Comparison of derived amino acid sequence of HUMMLC2B with database sequences revealed highly conserved 12 amino acid residues in a putative calcium-binding region. RT-PCR analysis showed a tissue-specific pattern of expression in skeletal muscle and a similar level of expression during skeletal muscle development. The possible role of HUMMLC2B and its relation to porcine heterosis are discussed.