We examined a possible mechanism to account for the maintenance of peak AMP deamination rate in fast-twitch muscle of rats fed the creatine analog -guanidinopropionic acid (-GPA), in spite of reduced abundance of the enzyme AMP deaminase (AMPD). AMPD enzymatic capacity (determined at saturating AMP concentration) and AMPD protein abundance (Western blot) were coordinately reduced ~80% in fast-twitch white gastrocnemius muscle by -GPA feeding over 7 wk. Kinetic analysis of AMPD in the soluble cell fraction demonstrated a single Michaelis-Menten constant (K_m; ~1.5 mM) in control muscle extracts. An additional high-affinity K_m (~0.03 mM) was revealed at low AMP concentrations in extracts of -GPA-treated muscle. The kinetic alteration in AMPD reflects increased molecular activity at low AMP concentrations; this could account for high rates of deamination in -GPA-treated muscle in situ, despite the loss of AMPD enzyme protein. The elimination of this kinetic effect by treatment of -GPA-treated muscle extracts with acid phosphatase in vitro suggests that phosphorylation is involved in the kinetic control of skeletal muscle AMPD in vivo.