The phosphorylated and unphosphorylated forms of tropomyosin Tpm1.1(α) are prepared from adult rabbit heart and compared biochemically. Electrophoresis confirms the high level of enrichment of the chromatography fractions and is consistent with a single site of phosphorylation. Covalently bound phosphate groups at position 283 of Tpm1.1(α) increase the rate of digestion at Leu-169, suggestive of a conformational rearrangement that extends to the midregion. Such a rearrangement, which is supported by ellipticity measurements between 25 and 42 °C, is consistent with a phosphorylation-mediated tightening of the interaction between various myofilament components. In a nonradioactive, co-sedimentation assay [30 mM KCl, 1 mM Mg(II), and 4 °C], phosphorylated Tpm1.1(α) displays a higher affinity for F-actin compared to that of the unphosphorylated control (Kd, 0.16 μM vs 0.26 μM). Phosphorylation decreases the concentration of thin filaments (pCa 4 plus ATP) required to attain a half-maximal rate of release of product from a pre-power stroke complex [myosin-S1-2-deoxy-3-O-(N-methylanthraniloyl)ADP-Pi], as investigated by double-mixing stopped-flow fluorescence, suggestive of a change in the proportion of active (turned on) and inactive (turned off) conformers, but similar maximum rates of product release are observed with either type of reconstituted thin filament. Phosphorylated thin filaments (pCa 4 and 8) display a higher affinity for myosin-S1(ADP) versus the control scenario without affecting isotherm steepness. Specific activities of ATP and Tpm1.1(α) are determined during an in vitro incubation of rat cardiac tissue [12 day-old, 50% phosphorylated Tpm1.1(α)] with [32P]orthophosphate. The incorporation of an isotope into tropomyosin lags behind that of ATP by a factor of approximately 10, indicating that transfer is a comparatively slow process.