Light spectroscopic properties of a recently developed fluorophore, 4,4-difluoro-4-borata-3a-azonia-4a-aza-s-indacene (BODIPY) and its derivatives, have been studied in different solvents, lipid membranes (modeled by lyotropic lamellar and cubic liquid crystals) as well as a protein. The absorption spectrum shows a strong {epsilon approximate to 90 000 M(-1) cm(-1)} S-0 --> S-1 transition at about 500 nm and a higher S-0 --> S-2 transition at about 375 nm, which is 20 times weaker. The electronic dipole of the S-0 --> S-1 transition is polarized along the long axis of the chromophore, as shown by linear dichroism spectroscopy (LD). The S-0 --> S-2 band contains a mixture of, presumably, in-plane-polarized transitions. For the S-0 --> S-1 transitions, the limiting fluorescence anisotropy, r(0) = 0.37. The fluorescence quantum yields are typically higher than 0.8. The calculated radiative lifetime of tau(0) = 5.6 ns in methanol is in reasonable agreement with the experimental value of tau(0) 5.9 +/- 0.2 ns. An efficient overlapping between the strong S-0 <-> S-1 transitions yields a Forster radius of R(0) = 57 +/- 1 Angstrom. The fluorescence lifetime and spectra are pH independent and change little with solvent polarity. The influence of oxygen quenching is typically less than 5% in liquids. The fluorescence of BODIPY is quenched by Trp (K = 15 M(-1)) and Tyr (K = 14 M(-1)). The quenching by Phe is less efficient, at least by one order of magnitude. The fluorescence relaxation of BODIPY is monoexponential when solubilized in lipid vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and a cubic liquid-crystalline phase of monoolein. The fluorescence decay of BODIPY, when covalently bound to the cystein residue of a mutant form of the plasminogen activator inhibitor 1 (PAI-1) is very well described by a sum of two exponential functions. The major component contributes with ca. 95% to the total intensity. In one of the PAI-1 mutants studied here, a Cys replaces the reactive center Ser 344 of the wildtype PAI-1. From the fluorescence anisotropy we conclude that the rotational correlation function of BODIPY in PAI-1 contains rapid motions (unresolved on the sub-ns time scales) and (at least) two rotational correlation times of about 3 and 50 ns. The longest correlation time is compatible with the tumbling motion of the protein molecule in water. Furthermore, it appears that the local rotational mobility of the BODIPY moiety in the reactive center of PAI-1 is restricted. The orientation and the rotational correlation function of different lipid derivatives of BODIPY in various lipid bilayers were studied by LD and time-resolved fluorescence spectroscopy.