Owing in part to their interactions with membrane proteins, polyamines (e.g., spermine, spermidine, and putrescine) have been identified as potential modulators of membrane excitability and Ca²⁺ homeostasis in cardiac myocytes. To investigate whether polyamines also affect cardiac myofilament proteins, we assessed the effects of polyamines on contractility using rat myocytes and trabeculae that had been permeabilized with Triton X-100. Spermine, spermidine, and putrescine reversibly increased the [Ca²⁺] required for half-maximal tension (i.e., right-shifted tension pCa curves), with the following order of efficacy: spermine (+4) > spermidine (+3) > putrescine (+2). However, synthetic analogs that differed from spermine in charge distribution were not as effective as spermine in altering isometric tension. None of the polyamines had a significant effect on maximal tension, except at high concentrations. After flash photolysis of DM-Nitrophen (a caged Ca²⁺ chelator), spermine accelerated the rate of tension development at low and intermediate but not high [Ca²⁺]. These results indicate that polyamines, especially spermine, interact with myofilament proteins to reduce apparent Ca²⁺ binding affinity and speed cross-bridge cycling kinetics at submaximal [Ca²⁺].