We investigated the ADP-ribosylation of AMP-deaminase from rat and rabbit skeletal muscle by guanidino compound-specific ADP-ribosyltransferase from hen liver nuclei and its effect on the enzyme activity. Rat AMP-deaminase was ADP-ribosylated when the enzyme was incubated with ADP-ribosyl-transferase and [adenylate-^32P] NAD. Preincubation of rabbit AMP-deaminase alone resulted in a loss of ADP-dependent, but not basal, enzyme activity. If, however, the enzyme was subjected to the ADP-ribosylation system containing unlabeled NAD and ADP-ribosyltransferase, the enzyme retained the property to be activated by ADP. Electrophoretic analyses of the enzyme preparation incubated with or without the ADP-ribosylation system and subsequently further incubation with trypsin showed that the ADP-ribosylation of the enzyme protects the enzyme from proteolysis concomitant with the retention of ADP-dependent activation of the enzyme.