Aminopeptidases (AP) are ubiquitously occuring, nonspecific exopeptidases involved in protein degradation. They cleave the N-terminal amino acid of peptides and occur in practically all mammalian cells and tissues. Physiological and pathological processes such as metastasis of tumors and inflammation have been thought to involve changes in AP activities. Determination of AP (EC 3.4.11.X) activity in viable cells by flow cytometry was the subject of this study because of its general biological and clinical interest. Dmerent bis-substituted rhodamine 110 (R110) peptide derivatives were synthesised and used as AP-and exopeptidase (EC 3.4.13.X-EC 3.4.14.X) substrates for flow cytometric measurements. Intracellular AP activities in viable lympho-, mono-, granulo-, and thrombocytes were detected by fluorescence increase from RllO following intracellular substrate cleavage. Eukaryotic-AP do not cleave D-aminoxids and hence NH,(D-Leu),RllO substrate served as negative control. Specifw substrate cleavage by AP is shown by complete inhibition of fluorescence generation following preincubation of cells with leucinechloromethylketone inhibitor. R l l O AP-and exopeptidase substrates are suitable indicators for coupled endopeptidase reactions due to their rapid cleavage and largely pH independent generation of intracellular fluorescence. o 1995 wiley-Liss, L ~C .