The Ca(2+)-dependent interaction of troponin I (TnI) with actin.tropomyosin (Tm) in muscle thin filaments is a critical step in the regulation of muscle contraction. Previous studies have suggested that, in the absence of Ca(2+), TnI interacts with Tm and actin in reconstituted muscle thin filaments, maintaining Tm at the outer domain of actin and blocking myosin-actin interaction. To obtain direct evidence for this Tm-TnI interaction, we performed photochemical crosslinking studies using Tm labeled with 4-maleimidobenzophenone at position 146 or 174 (Tm*146 or Tm*174, respectively), reconstituted with actin and troponin [composed of TnI, troponin T (TnT), and troponin C] or with actin and TnI. After near-UV irradiation, SDS gels of the Tm*146-containing thin filament showed three new high-molecular-weight bands determined to be crosslinked products Tm*146-TnI, Tm*146-troponin C, and Tm*146-TnT using fluorescence-labeled TnI, mass spectrometry, and Western blot analysis. While Tm*146-TnI was produced only in the absence of Ca(2+), the production of other crosslinked species did not show Ca(2+) dependence. Tm*174 mainly crosslinked to TnT. In the absence of actin, a similar crosslinking pattern was obtained with a much lower yield. A tryptic peptide from Tm*146-TnI with a molecular mass of 2601.2 Da that was not present in the tryptic peptides of Tm*146 or TnI was identified using HPLC and matrix-assisted laser desorption/ionization time-of-flight. This was shown, using absorption and fluorescence spectroscopy, to be the 4-maleimidobenzophenone-labeled peptide from Tm crosslinked to TnI peptide 157-163. These data, which show that a region in the C-terminal domain of TnI interacts with Tm in the absence of Ca(2+), support the hypothesis that a TnI-Tm interaction maintains Tm at the outer domain of actin and will help efforts to localize troponin in actin.Tm muscle thin filaments.