We have used rapid mixing negative stain electron microscopy (EM) to observe myosin heads (S1) bound to F-actin and thin filaments in the presence of ATP. Myosin V S1 was mixed with excess ATP, incubated for 2 sec to allow binding and hydrolysis, and then mixed with either F-actin or cardiac thin filaments. This mixture was applied to an EM grid and negatively stained with uranyl acetate ∼20 ms later. Short times on the grid allow protein concentrations as high as 10 uM to be used, which is critical to observe weakly binding complexes, such as S1-ADP-Pi with actin filaments. This approach has advantages compared to hand mixed solutions. It has improved time resolution (0.3 s vs 3 s between mixing and staining), the mixing is ∼1000x faster compared to manual manipulation (∼ 2 ms vs 2 s) and mixing is more uniform. The entire automated procedure used an apparatus with computer controlled stepper motors previously built for time-resolved cryo-EM (White et al, J Struct Biol:144, 246-52, 2003), which was modified for negative staining. Slow and non-uniform mixing produced by hand mixing can suggest cooperative binding. After automated mixing, S1-ADP-Pi with cardiac thin filaments at low calcium concentrations, S1 bound randomly and only rarely were bare or fully decorated filaments observed. Without ATP, thin filaments were mostly either bare or fully decorated, suggestive of cooperativity. These results may explain the apparently contradictory observations that binding of S1 to thin filaments appeared to be cooperative in micrographs, whereas kinetic and structural measurements indicate that individual rigor heads activate 12-14 actin subunits.