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Methods for using synthetic peptides to specifically probe the molecular mechanisms for calcium-dependent regulation of contraction in cardiac and smooth permeabilized (or skinned) muscle are described. As examples of the use of these tools, the role of troponin in modulating the cardiac crossbridge cycle and the regulatory action of myosin light chain kinase (MLCK) in smooth muscle in Triton X-100-extracted muscle preparations have been targeted. These "skinned" fibers are functional in terms of contractility but permit precise control of aspects of the "cytoplasmic" environment around the myofilaments, such as calcium and substrate concentration. They also permit the diffusion of peptides into the ′intracellular" compartment. These include peptides derived from the common actin-binding, troponin C-binding sequence of troponin I (the so-called inhibitory sequence, Tnl 104–115) and the calmodulin-binding sequence of MLCK (also known as RS20). The effects of these peptides were monitored in terms of changes in isometric tension and expressed as changes in calcium or calmodulin sensitivity. The calmodulin-binding peptide reduced force at a fixed calcium concentration, indicating decreased calcium sensitivity. This effect was associated with a moderate decrease in myosin light chain phosphorylation and could be reversed with increased calmodulin concentration. We interpret this latter observation to mean that underlying the change in apparent calcium sensitivity is a change in the sensitivity of MLCK to calmodulin. As previously reported, the troponin I-based peptide desensitizes skinned cardiac muscle with respect to calcium by inhibiting the actin activation of the crossbridge cycle. We also discuss the results of recent experiments in which this peptide was used in conjunction with a calcium-sensitizing compound, EMD 53998. These results implicate the phosphate release step as the most likely regulatory step in the crossbridge cycle affected by the peptide and, by extension, troponin I. Peptide studies such as these have provided useful specific insights into the highly complex and multivariable regulatory systems of contraction.