Novel fluorescent probes of the generic formula Ar-(CH2)(n)-NH3+ (Scheme I, Ar = 9-anthryl or 3-pyrenyl) were synthesized, and their binding properties with bovine serum albumin (BSA) have been evaluated. The anthryl probes 9-anthrylmethylamine hydrochloride (AMAC), N-ethyl(9-anthryl)methylamine hydrochloride (N-Et-AMAC),and 3-(9-anthryl)propylamine hydrochloride (APAC) showed small changes in their absorption spectra upon binding to BSA, whereas the pyrenyl analog, 4-(1-pyrenyl)butylamine hydrochloride (PBAC), showed a 5-nm red shift and an increase in the extinction coefficient at the peak positions. Such a red shift and increase in the intensity of the absorption transitions are consistent with binding of PBAC to hydrophobic sites on the protein. The fluorescence spectra of the anthryl and pyrenyl analogs exhibit different trends. The anthryl analog emission was quenched very effectively by increasing amounts of BSA (K-sv similar to 2.6 X 10(3) M(-1)). In contrast, PBAC emission was quenched at low BSA concentration whereas at higher concentrations of the protein the emission was enhanced. The fluorescence decays of the anthryl probes bound to the protein can be described by a short-lived and a long-lived component (10.6 and 6.7 ns for AMAC, 10.3 and 5.1 ns for APAC, 14.6 and 7.8 ns for N-Et-AMAC) indicative of at least two types of binding sites. In the case of PBAC, a third component was observed at probe:protein ratios higher than 1:5, which may be due to an exciplex formed at the binding site. Data from the equilibrium dialysis experiments indicate that the order of protein binding affinity of these probes is PBAC >> APAC > N-Et-AMAC > AMAC. Steady-state and time-resolved fluorescence quenching experiments with potassium iodide confirmed the above trend in the binding affinities. Upon binding to BSA, APAC and PBAC emission was extensively protected whereas only moderate protection has been observed for N-Et-AMAC and AMAC. A comparison of the binding properties of AMAC and N-Et-AMAC shows that increased distance of separation between the hydrophobic moiety and the cationic function enhances the protein binding affinity. Additionally, comparison of APAC with PBAC revealed the strong role of hydrophobic groups in the binding interactions. Therefore, the protein binding affinity of these probes depends on the degree of hydrophobicity of the aromatic moiety and on the length of the linker separating the hydrophobic group the cationic function.