The activity of AMP-activated protein kinase (AMPK) increases during muscle contractions as a result of elevated AMP concentration. We tested whether activation of AMPK would be altered during contractions in adenylate kinase 1-deficient (AK1^-/-) mice, as they have a reduced capacity to form AMP. The right gastrocnemius-soleus-plantaris muscle group was stimulated via the sciatic nerve at 2 Hz for 30 min in both wild-type (WT) and AK1^-/- animals. Initial force production was not different between the two groups (129.2 ± 3.3 g vs. 140.9 ± 8.5 g for WT and AK1^-/-, respectively); however, force production by AK1^-/- mice was significantly greater over the 30 min stimulation period and final tension was 85 ± 4.5% of initial in WT and 102 ± 3.2% initial in AK1^-/- mice. Western blot analysis showed that AMPK phosphorylation with contractions was clearly increased in WT muscles (4.0± 1.1 above resting values), but did not change noticeably with AK deficiency (1.6 ± 0.4 above WT resting values). However, increases in phosphorylation of acetyl CoA carboxylase (ACC) were robust in both WT and AK1^-/- muscles and not different between the two groups. These results suggest that reduced formation of AMP during contractions in skeletal muscle of AK1^-/- mice results in reduced phosphorylation of AMPK. However, altered AMPK signaling was not apparent in the phosphorylation status of ACC, a typical marker of AMPK activity.