Porcine congenital splayleg (PCS) is the most important congenital condition of piglets, associated with lameness and immobility, of unknown aetiology and pathogenesis. The aim of this study is to investigate the cellular and molecular changes in skeletal muscles of PCS, thereby gaining new molecular insights into this clinical condition. Based on immunohistochemistry and histological image analyses on 4 sets of 2-day- old splayleg piglets, each with a corresponding normal litter mate, a consistent discovery has been that PCS muscles [semitendinosus (ST), longissimus dorsi (LD) and gastrocnemius (G)] showed extensive fibre atrophy without apparent tissue damage. At present, it is not certain if PCS-associated fibre atrophy is accompanied by fibre hypoplasia. Both normal and PCS muscle fibres showed similar widespread distribution of lipid- and oxidative-positive fibres. Although there was no significant difference in fibre type composition, several structural myosin heavy chain (MyHC) genes were significantly down-regulated in PCS affected muscles. Interestingly, MAFbx, a major atrophy marker, was highly up-regulated in almost all PCS muscles, when compared with controls from normal litter mates. In contrast, P311, a novel 8- kDa protein, was relatively down-regulated in all PCS muscles examined. To further investigate the functional role of P311 in skeletal muscle, its full-length cDNA was sequenced (Accession. N0. EF416570) and over-expressed in murine C2C12 muscle cells. P311 over-expression enhanced cell proliferation and reduced myotube formation in C2C12 cells. The over-expression of calcineurin, a key intracellular calcium-dependent signalling factor of muscle differentiation, down- regulated P311 expression. Reduced P311 expression in PCS piglets might contribute to atrophy through reduced myotube contribution. To investigate the functional role of SPARCL-1, a matricellular secreted glycoprotein that belongs to SPARC family, its full-length cDNA was sequenced (Accession. N0. EF416571) and over-expressed in murine C2C12 muscle cells. SPARCL-1 overexpression led to reduced cell proliferation and down-regulation of MyHC genes during late differentiation. SPARCL-1 might be associated as a negative regulator of skeletal muscle cell proliferation and cell differentiation. However, endogenous SPARCL-1 expression was similar between PCS and normal muscles. Hence, although SPARCL-1 could play a role in muscle development, it is unlikely to be a main factor in the development of PCS. In summary, PCS is shown to be a condition characterised by extensive fibre atrophy and raised fibre density, and it is proposed that the combined differential expression of MAFbx and P311 is of potential value in the diagnosis of sub-clinical PCS.