Two-photon-excited fluorescence and second-harmonic generation are characterized as a function of laser pulse duration as short as sub-. A comparative study is performed where pulse duration is varied by introducing dispersion, as reported previously, and by tailoring pulse spectral width and minimizing its time-bandwidth product (transform-limited pulses). Experimental data and calculations show that characterizing a two-photon signal with the two schemes to vary pulse duration measures different phenomena. Two-photon signal characterization using dispersion-broadened pulses measures only the effect of chirp on the pulse two-photon-excitation spectrum and is independent of molecular response. Transform-limited pulses are used to measure the dependence of two-photon signal generation on pulse duration. Calculations show that deviation from the relationship would be expected as the transform-limited pulse spectral width approaches the molecular two-photon absorption linewidth and exhibits asymptotic behavior for pulse spectral widths 10 times greater than the absorption linewidth. Experimental measurements are consistent with the predicted behavior. The impact of using ultrashort laser pulses on the performance characteristics of nonlinear optical microscopy is discussed.