Delayed extraction experiments were undertaken to gain a better insight into the dynamic effects involved in the ion formation in UV matrix‐assisted laser desorption/ionization. Part I1 was devoted to a 2,5‐dihydroxybenzoic (2,5‐DHB) matrix. The results clearly demonstrated the existence and the role of high‐mass precursors corresponding to a non‐covalent matrix–analyte association in ion formation. In this complementary study, ion flight time and abundance were studied as a function of the delay extraction time using the matrix α‐cyano‐4‐hydroxycinnamic acid (HCCA). Under our instrumental conditions, where ejected ions experienced a low repulsing electric field before extraction, two main results were obtained: (i) two ion components are observed in the peak profiles depending on the repulsing field, a first, major component (I) similar to that observed for 2,5‐DHB and a second, minor component (II) apparently triggered by the delayed extraction pulse, and (ii) ion time‐of‐flight variation vs delay time remained lower than that noted with 2,5‐DHB matrix, indicating that the initial axial velocity is smaller. The initial kinetic energy of matrix and low molecular mass peptide ions for the component I is not high enough to overcome the repulsing potential in the delay time range (200–2200 ns) and we have to assume that ions have non‐covalent clusters as precursors. Complete desolvation of these clusters–aggregates would be achieved through the extraction step. Simulations of the ion time‐of‐flight as a function of the delay time allow the determination of the average size of the precursors, typically 4500, 40000 and 50000 u for HCCA, ACTH 7–38 and bovine insulin quasi‐molecular ion, respectively, assuming that the precursors are singly charged. The size of these ion precursors is greater than that of those generated for 2,5‐DHB. For component II, ions are probably not solvated and they are directly desorbed from the target. Taking into account the results on HCCA and 2,5‐DHB matrices and other results from the literature, a general model for ion formation based on clusters as ion precursors is proposed and discussed. Copyright © 2004 John Wiley & Sons, Ltd.