To study differential gene expression in porcine skeletal muscle, a porcine complementary DNA (cDNA) macroarray was produced that contained 327 expressed sequence tags (EST) derived from whole embryo and adult skeletal muscle, and differential display PCR products from fetal and postnatal muscle. Total RNA from four muscle samples, 75- and 105-d fetal hind limb muscles, and 1- and 7-wk postnatal semitendinosus muscle was used to make radiolabeled targets for duplicate hybridization to the macroarray membranes in an initial screen for expression. All EST that gave clear signals (n = 238) were then re-arrayed, and hybridization was conducted with additional biological replication of samples in the 75-d and 1-wk ages. Signal intensity for each gene was normalized to signal intensity measured at control spots on each membrane, which consisted of total cDNA from liver, lung, spleen, and skeletal muscle. Both normalized ratio levels and a mixed linear model analyses were used to identify genes differentially expressed among the muscle samples. Results showed 28 genes had differences in expression level greater than twofold between the 75-d fetal and 1-wk muscle RNA samples. All 28 genes were also identified as genes with significantly different (P < 0.01) expression using a mixed linear model analysis. Nineteen of these 28 genes had significant matches (basic local alignment search tool [BLAST] score > 100; P < 0.01) to known genes, two matched genes encoding human hypothetical proteins, and seven had no significant matches to Genbank nonredundant and dbEST (database of expressed sequence tags) entries. These results were confirmed for representative genes with RNA blot analysis of seven developmental time points, including RNA from the same muscle samples tested previously in the macroarray. The RNA blot results confirmed the macroarray results for all selected genes, demonstrating that the macroarray technique used in this study is accurate and reproducible. An unknown muscle clone (M218) with a slightly less than twofold increase in expression from the 75-d to the 1-wk age (1 wk/75 d = 1.94; P = 0.0114) was also shown to differ between these two ages using RNA blot analysis, demonstrating the methods used to identify differentially expressed genes may be conservative. The association between expression patterns of vimentin and desmin was also investigated. Results indicate the switch in intermediate filament protein from vimentin to desmin occurs primarily at the level of transcription and/or RNA processing.