The unfolding dynamics of a 16 amino acid peptide (Ac-EACAREAAAREAACRQ-NH(2), FK-11-X) was followed using nanosecond time-resolved optical rotatory dispersion (ORD). The peptide was coupled to an azobenzene linker that undergoes subnanosecond photoisomerization and reisomerizes on a time scale of minutes. When the linker is in the trans form, the peptide favors a more helical structure (66% helix/34% disordered) and when in the cis configuration the helical content is reduced. Unfolding of FK-11-X was rapidly triggered by a 7-ns laser pulse at 355 nm, forming cis azobenzene-linked peptides that maintained the secondary structure (helical or disordered) of their trans azobenzene counterparts. The incompatibility of the instantaneous cis photoproduct with helical secondary structure drives the subsequent peptide unfolding to a new conformational equilibrium between cis helix and cis disordered structures. The kinetic results show a approximately 40% decrease in the time-dependent ORD signal at 230 nm that is best fit to a single-exponential decay with a time constant of 55 +/- 6 ns. Folding and unfolding rates for cis FK-11-X are estimated to be approximately 3.0 x 10(6) s(-)(1) (1/330 ns) and approximately 1.5 x 10(7) s(-)(1) (1/66 ns), respectively.