The kinetics of guest ion formation in matrix-assisted laser desorption ionization was studied for substance P and insulin from 2,5-dihydroxybenzoic acid (DHB), alpha-cyano-4-hydroxycinnamic acid (CHCA), 9-anthracenecarboxylic acid (9-ACA), and sinapinic acid (SA) matrixes. The results show two very different responses. Although guest ion yields from DHB and SA follow a simple fast exponential decay (with decay times of 4.3 +/- 0.7 ns and 4.6 +/- 0.8 ns, respectively), the ions from the other two matrixes show extended maxima (at 6.1 ns for CHCA and 3.2 ns for 9-ACA) with only a moderate decline in their yield. Possible explanations for the fast exponential decay include the relaxation of electronically and/or vibrationally excited matrix precursors for DHB and SA. The explanation of extended maxima in the case of 9-ACA and especially CHCA can be based on competition for the protons between matrix and guest species. CHCA has significantly lower proton affinity than SA, thus, resulting in lower deprotonation rates for guest ions. There is, however, an alternative explanation. As long as the matrix surface temperature exceeds the sublimation temperature, there is sufficient vaporization to sustain a dense MALDI plume and facilitate guest ionization.