Under nearly identical conditions, the difference between 500-fs and 5-ns laser pulse ionization and dissociation of small volatile molecules and large laser-desorbed polypeptides in the gas phase is demonstrated. By applying in each experimental cycle almost simultaneously but independently both femtosecond and nanosecond laser pulses for photoionization, reproducible intensity relations in the mass spectra have been achieved even for laser-desorbed molecules. For femtosecond and nanosecond laser pulse excitation, a large laser intensity difference results for equal laser pulse energy. Nevertheless, we observe the ion yield to be nearly the same for small molecules. For large molecules femtosecond excitation is found to be much more efficient than nanosecond excitation. We explain this by fast relaxation rates which can compete either with photon absorption or with ionization. The fragmentation of large ions demonstrates a most important difference between the two modes of excitation. Femtosecond excitation shows large fragmentation near the photoexcited chromophores, while nanosecond excitation produces statistical energy redistribution and thus a lot of different fragments. This lack or presence of internal energy randomization in the same molecular system has consequences for future applications in site-specific bond-breaking processes and for the unimolecular rate theory of very large molecular systems.