Chromogenic and fluorogenic substrates are useful tools for the determination of the activity of isolated enzymes. Unfortunately, the quantification of enzymatic activity in cellular systems using these compounds is limited. A maior obstacle is the diffusion of the enzymatically released fluorophore resulting in a high background fluorescence complicating the detection of enzymatic activity on cells. The present work deals with the synthesis and the spectroscopic and enzymkinetic investigation of rhodamine 110(R110)-based substrates for the prolinespecific serine protease dipeptidylpeptidase IV (DP IV). Based upon compounds of the structure (Xaa-Pro)2-R110, the suitability of these substrates for the detection of activity of isolated as well as cell-associated DP IV was studied. The advantage of these compounds is the high sensitivity of the fluorophore R110. To prevent the diffusion of the hydrolysis product R110 from the cells and thereby to improve the quantification of enzymatic activity especially in cellular systems, bifunctional compounds Xaa-Pro-R110-Y (Xaa = Gly or Ala) were synthesized. The residue Y represents an anchor structure with different hydrophobicity, reactivity and length (Y = halogenalkylcarbonyl-, halogenalkylarylcarbonyl- and maleinimidoalkylcarbonyl-residues). By these reactive anchor groups a stable fixation of the fluorophore on the cell surface was realized. This way, the microscopic differentiation of cells differing in their DP IV activity comparable to the differentiation via antiDP IV antibody staining was possible. The application of these compounds for the detection of DP IV activity using the flow cytometry is limited. In this regard different biological effects as possible reasons for the limited differentiation of DP IV activity on mononuclear blood lymphocytes are discussed. Beside fluorogenic substrates specific inhibitors of DP IV, Nε-4(5)-carboxyfluoresceine- and biotin-labeled Lys-2-cyanopyrrolidides, were synthesized and enzymatically characterized. The suitability of these compounds for the localization of cellular DP IV was studied.