Tropomyosin (Tm) binds along actin filaments, one molecule spanning four to seven actin monomers, depending on the isoform. Periodic repeats in the sequence have been proposed to correspond to actin binding sites. To learn the functional importance of length and the internal periods we made a series of progressively shorter Tms, deleting from two up to six of the internal periods from rat striated alpha-TM (dAc2--3, dAc2--4, dAc3--5, dAc2--5, dAc2--6, dAc1.5--6.5). Recombinant Tms (unacetylated) were expressed in Escherichia coli. Tropomyosins that are four or more periods long (dAc2--3, dAc2--4, and dAc3--5) bound well to F-actin with troponin (Tn). dAc2--5 bound weakly (with EGTA) and binding of shorter mutants was undetectable in any condition. Myosin S1-induced binding of Tm to actin in the tight Tm-binding "open" state did not correlate with actin binding. dAc3--5 and dAc2--5 did not bind to actin even when the filament was saturated with S1. In contrast, dAc2--3 and dAc2--4 did, like wild-type-Tm, requiring about 3 mol of S1/mol of Tm for half-maximal binding. The results show the critical importance of period 5 (residues 166--207) for myosin S1-induced binding. The Tms that bound to actin (dAc2--3, dAc2--4, and dAc3--5) all fully inhibited the actomyosin ATPase (+Tn) in EGTA. In the presence of Ca(2+), relief of inhibition by these Tms was incomplete. We conclude (1) four or more actin periods are required for Tm to bind to actin with reasonable affinity and (2) that the structural requirements of Tm for the transition of the regulated filament from the blocked-to-closed/open (relief of inhibition by Ca(2+)) and the closed-to-open states (strong Tm binding to actin-S1) are different.