Mono-ADP-ribosylation is the enzymatic transfer of ADP-ribose from $NAD^+$ to acceptor proteins catalyzed by ADP-ribosyltransferases. Using m-aminophenylboronate affinity chromatography, 2D-gel electrophoresis, in-gel digestion and MALDI-TOF analysis we have identified eight in vitro ADP-ribosylated proteins in Streptomyces coelicolor, which can be classified into three categories: (i) secreted proteins; (ii) metabolic enzymes using $NAD^+$/NADH or $NADP^+$/NADPH as coenzymes; and (iii) other proteins. The secreted proteins could be classified into two functional categories: SCO2008 and SCO5477 encode members of the family of periplasmic extracellular solute-binding proteins, and SCO6108 and SCO1968 are secreted hydrolases. Dehydrogenases are encoded by SCO4824 and SCO4771. The other targets are GlnA (glutamine synthetase I., SCO2198) and SpaA (starvation-sensing protein encoded by SCO7629). SCO2008 protein aod GlnA had been identified as ADP-ribosylated proteins in previous studies. With these results we provided experimental support for a previous suggestion that ADP-ribosylation may regulate membrane transport and localization of periplasmic proteins. Since ADP-ribosylation results in inactivation of the target protein, ADP-ribosylation of dehydrogenases might modulate crucial primary metabolic pathways in Streptomyces. Several of the proteins identified here could provide a strong connection between protein ADP-ribosylation and the regulation of morphological differentiation in S. coelicolor.