The tropomodulin (Tmod) family of proteins that cap the pointed, slow-growing end of actin filaments require tropomyosin (TM) for optimal function. Earlier studies identified two regions in Tmod1 that bind the N terminus of TM, though the ability of different isoforms to bind the two sites is controversial. We used model peptides to determine the affinity and define the specificity of the highly conserved N termini of three short, non-muscle TMs (α, γ, δ-TM) for the two Tmod1 binding sites using circular dichroism spectroscopy, native gel electrophoresis, and chemical crosslinking. All TM peptides have high affinity for the second Tmod1 binding site (within residues 109–144; α-TM, 2.5 nM; γ-TM, δ-TM, 40–90 nM), but differ >100-fold for the first site (residues 1–38; α-TM, 90 nM; undetectable at 10 μM, γ-TM, δ-TM). Residue 14 (R in α; Q in γ and δ) and, to a lesser extent, residue 4 (S in α; T in γ and δ) are primarily responsible for the differences. The functional consequence of the sequence differences is reflected in more effective inhibition of actin filament elongation by full-length α-TMs than γ-TM in the presence of Tmod1. The binding sites of the two Tmod1 peptides on a model TM peptide differ, as defined by comparing ¹⁵N,¹H HSQC spectra of a ¹⁵N-labeled model TM peptide in both the absence and presence of Tmod1 peptide. The NMR and CD studies show that there is an increase in α-helix upon Tmod1–TM complex formation, indicating that intrinsically disordered regions of the two proteins become ordered upon binding. A model proposed for the binding of Tmod to actin and TM at the pointed end of the filament shows how the Tmod–TM accentuates the asymmetry of the pointed end and suggests how subtle differences among TM isoforms may modulate actin filament dynamics.