During postnatal growth and after muscle injury, satellite cells proliferate and differentiate into myotubes to form and repair musculature. Comparison of studies on satellite cell proliferation and differentiation characteristics is confounded by the heterogeneity of the experimental conditions used. To examine the influence of sex, age, and fiber-type origin on in vitro properties of satellite cells derived from postnatal muscles, fast extensor digitorum longus (EDL) and slow soleus (SOL) muscles were extracted from male and female mice of 1 week to 3 months of age. Myoblast proliferation and myogenic regulatory factor (MRF) expression was measured from cultures of freshly isolated satellite cells. Higher proliferation rate and elevated Myod1 expression was found in male EDL and SOL derived cells compared with females at age of 40, 60, and 120 days, whereas inverse tendency for cell proliferation was apparent in EDL of juvenile (7-day-old) pups. Myogenin and Mrf4 transcripts were generally elevated in males of 40 and 60 days of age and in female EDL of juveniles. However, these differentiation markers did not significantly correlate with proliferation rate at all ages. Pax7, whose overexpression can block myogenesis, was up-regulated especially in 40-day-old females where MRF expression was low. These results indicate that gender, postnatal age, and muscle fiber origin affect proliferation and muscle transcription factor expression in vitro. The results also support the view that satellite cells originating from slow and fast muscles are intrinsically different and warrant further studies on the effect of cell origin for therapeutic approaches. J. Cell. Biochem. 112: 2825–2836, 2011. © 2011 Wiley-Liss, Inc.