2-pyridone (2Py) and 3-pyridone (3Py) were examined in different solvents and their binding to human serum albumin (HSA) was studied using steady-state spectroscopy and time-resolved fluorescence. Solvation of 2Py and 3Py by water was examined in binary mixtures of 1,4-dioxane and water. Analysis of the absorption and fluorescence data reveals the solvation of the hydrogen bonding center in 2Py by one water molecule and in 3Py by three water molecules. A zwitterionic tautomer of 3Py is formed in water and shows distinct absorption peaks from the absorption of the neutral tautomer. Fluorescence of 3Py was observed in polar solvents only, whereas 2Py is fluorescent in polar and nonpolar solvents. The absorption and fluorescence spectra of 2Py in different solvents indicate less solute-solvent interaction in nonpolar solvents. This observation was confirmed by the measured longer fluorescence lifetime of 2Py in cyclohexane compared to that in water. The mechanism of binding of 2Py and 3Py as probe ligands to HSA was investigated by following the intensity change and lifetime of HSA fluorescence after excitation at 280 nm. The presence of 2Py and 3Py causes a reduction in the fluorescence intensity and lifetime of HSA. This observation indicates that subdomain IIA binding site (Sudlow site I) is the host of the probes and the reduction in the fluorescence of HSA is due to energy transfer from the Trp-214 residue to the probe in each case. The distance between Trp-214 and each of the probes was calculated using Förster theory for energy transfer to be 1.99 nm for HSA/2Py and 2.44 nm for HSA/3Py. The shorter distance in the former complex indicates more efficient energy transfer than in the latter. This was confirmed by estimating the quenching rate constant (kq) in each complex. kq was calculated to be 1.44 × 1012 M-1S-1 for HSA/2Py and 3.45 × 1011 M-1 s-1 for HSA/3Py. The calculated distances and the kq values indicate a static quenching mechanism operative in the two complexes. The binding constants were estimated to be K = (3.4 ± 0.4) × 104 M-1 for the HSA/2Py complex and K= (2.3 ± 0.3) × 104 M-1 for the HSA/3Py complex. The number of binding sites of HSA was calculated to be one in both complexes. The latter results, along with the quenching results, indicate that both probes, 2Py and 3Py, bind only in Sudlow site I in subdomain IIA.