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It was proposed from cellular studies thatS. pombetropomyosin Cdc8 (Tpm) segregates into two populations due to the presence or absence of an amino-terminal acetylation that specifies which formin-mediated F-actin networks it binds, but with no supporting biochemistry. To address this mechanismin vitro, we developed methods forS. pombeactin expression in Sf9 cells. We then employed 3-color TIRF microscopy using all recombinantS. pombeproteins to probein vitromulticomponent mechanisms involving actin, acetylated and unacetylated Tpm, formins, and myosins. Acetyl-Tpm exhibits tight binding to actin in contrast to weaker binding by unacetylated Tpm. In disagreement with the differential recruitment model, Tpm showed no preferential binding to filaments assembled by the FH1-FH2-domains of twoS. pombeformins, nor did Tpm binding have any bias towards the growing formin-bound actin filament barbed end. Although ourin vitrofindings do not support a direct formin-tropomyosin interaction, it is possible that formins bias differential tropomyosin isoform recruitment through undiscovered mechanisms. Importantly, despite a 12% sequence divergence between skeletal andS. pombeactin,S. pombemyosins Myo2 and Myo51 exhibited similar motile behavior with these two actins, validating key prior findings with these myosins that used skeletal actin.