Skeletal muscle is unique in its growth and developmental characteristics in that normal hypertrophy is carried out through activation and fusion of satellite cells (SC) surrounding individual muscle fibers. These SC are responsive to signals from their extracellular environment especially growth factors and nutrients. Polyamines (PA) are a class of small, positively charged molecules that favorably affect growing cells through somewhat undefined physiological mechanisms. Polyamines exist in mammalian species primarily as ornithine derivatives. Abnormalities in polyamine metabolism are associated with irregular development in mammals emphasizing their critical role as regulators of growth. To test the direct effect of PA on skeletal muscle development, primary bovine SC cultures were differentiated in media containing either methionine (10 mM control 1), ornithine (10 mM), putrescine (5 mM), spermine (0.5 mM), or no supplement (control 2). SC were differentiated as a monoculture (MC) as well as in coculture (CC) alongside preadipocytes isolated from bovine intramuscular fat depots. This coculture provided a more realistic environment of that which would surround skeletal muscle and impact SC activity. Following treatment, SC were isolated from CC using laser micro-dissection technique. SC populations were analyzed for temporal expression of the myogenic regulatory factors (MRF) MyoD, Myf5, and Myf4 (myogenin) to identify differentiating cells along with the genes Pax7 and Spry1 representative of quiescent cells. Protein isolation and Western Blot analyses were also performed to measure protein expression in a temporal manner. Exposure of SC’s to PA in CC resulted in upregulation of MyoD (P = 0.05), Myf5 (P = 0.02), and Myf4 (P = 0.09) and attenuation of Pax7 (P = 0.10) and Spry1 (P = 0.07). Statistics generated in SAS (Cary, NC) using PROC MIXED procedure. Treatment was included as a fixed effect. These results suggest that ornithine metabolism and polyamine metabolites can affect bovine skeletal muscle myogenesis, and may therefore be promising as candidates for natural means of promoting growth of lean tissue in cattle.