Reduced plasticity of senescent skeletal muscle may be due to an attenuated signaling response to contractions. The intent of the present study was to determine whether aging affects signaling pathways involved in mitochondrial biogenesis in response to a single bout of contractile activity (CA). Acute stimulation (1 Hz, 5 min) of the tibialis anterior (TA) resulted in a significantly greater rate of fatigue in 36 month (mo) relative to 6 mo F344XBN rats. To investigate fiber type-specific signaling, the TA was sectioned into red (RTA) and white (WTA) portions, possessing 2.5-fold differences in mitochondrial content. The CA-mediated activation of p38, MKK3/6, CaMKII and AMPK signaling was assessed. In general, CA-evoked kinase phosphorylation was greater in RTA compared to WTA in both age groups. p38 activation was similar in RTA from 6 and 36 mo groups, however in WTA, the response was 2-fold greater in muscle from 6, relative to 36 mo animals. CA also increased MKK3/6 phosphorylation, the upstream regulator of p38, however the magnitude was 70–80% higher in RTA and WTA of 6, compared to 36 mo rats. Contraction-induced CaMKII phosphorylation was 40% higher in both fiber types in muscle from 6 relative to 36 mo animals. AMPK was not responsive to 1 Hz CA at either age. Thus, CA-induced signaling to mitochondrial biogenesis is reduced in aging muscle, but the extent of the attenuation depends in part on mitochondrial content.