The antiparallel dimer (APD) is a unique actin species, which can be detected in the early stages of actin polymerization. In this work, we introduce novel tools for examination of the effects of the APD on actin polymerization. We document that bifunctional methanothiosulfonate (MTS) reagents are an attractive alternative to the routinely used p-phenylene maleimide (pPDM) for APD detection, allowing for fast and efficient cross-linking under conditions of actin polymerization at neutral pH. We report also that pyrene-labeled yeast actin mutant A167C/C374A (C167PM) forms significant amounts of stable APD in solution, without chemical cross-linking or polymerization-affecting compounds, and that the kinetics of APD transformation and decay upon actin polymerization can be easily monitored. The dimerization of C167PM has been characterized in sedimentation equilibrium experiments (K(d) approximately 0.3 microM). This new system offers the advantage of assessing the effects of the APD under physiological conditions (pH, ionic strength, and Mg(2+) concentration) and testing for conformational transitions in the APD during nucleation-polymerization reactions or/and in the presence of actin-interacting factors. The results obtained using two different systems (C167PM actin and polylysine-induced polymerization of alpha-actin) show that the APD decays at a rate slower than that at which the filaments elongate, revealing its transient incorporation into filaments, and confirm that it inhibits the nucleation and elongation of actin filaments.