The photophysical properties of the fluorescence energy-transfer donor, 2-aminobenzamide (Abz), and nitrotyrosine (Tyr-NO2) acceptor have been evaluated. The 31-Angstrom Forster radius for this donor-acceptor pair makes it useful for evaluating molecular separations in the range of 15-45 Angstrom. Abz and Tyr-NO2 have been incorporated into four medium-sized synthetic peptides (1, Abz-SEEEEKKKKEEEEKKKK(Tyr(NO2))D; 2, Abz-AEAAAKHAAAHEAAAKA(Tyr(NO2))D; 3, Abz-FAQKEPAFLKEYHLL(Tyr(NO2))D; 4, Abz-LKELKDKLKELKDKLK(Tyr(NO2))LKD). Nonexponential Abz fluorescence decays indicate that the synthetic peptides do not assume single, static conformations in solution. The fluorescence decay kinetics in three of the four peptides have been fit to a model in which the peptide conformations are described by Gaussian distributions of donor-acceptor distances centered at r(C) (r(C) (fwhm) : 2, 27.0 (8.1); 3, 25.6 (12.9); 4, 21.1 (22.1) Angstrom). Values of r(C) are in good agreement with distances obtained from molecular dynamics simulations (r(MD) : 2, 28; 3, 21; 4, 22 Angstrom).