Thromboxane A2 synthase (TXAS) has been proposed to have two membrane-bound regions located in the NH2-terminal domain [Ruan, K.-H., Wang, L.-H., Wu, K. K., and Kulmacz, R. J. (1993) J. Biol. Chem, 268, 19483-19489; Ruan, K.-H., Li, P., Kulmacz, J. R., and Wu, K. K. (1994) J. Biol. Chem, 269, 20938-20942]. To test this hypothesis, a solution structure in membrane mimetic environments of a synthetic peptide corresponding to the second region of the NH2-terminal domain (TXAS residues 33-60) has been investigated by circular dichroism (CD), 2D nuclear magnetic resonance (NMR) spectroscopy, and peptidoliposome reconstitution. CD spectroscopy indicated that the peptide adopted a structure with significant alpha-helical content in 30% trifluoroethanol (TFE) or in dodecylphosphocholine (DPC) micelles, which mimic hydrophobic membrane environment. Through a combination of 2D NMR experiments in the presence of TFE or DPC micelles, complete 1H NMR assignments of the peptide have been obtained and the structure of the peptide has been determined. NH2-terminal segment of the peptide takes on a well-defined alpha-helical conformation; the center segment of the peptide, containing three prolines, adopts a bent conformation, and the C-terminal segment of the peptide exists in a mixture of rapidly interconverting conformations. These results provide direct structural evidence that residues 33-60 of the TXAS NH2-terminal domain contain a second membrane anchor region, with at least residues 35-46 having their helical structure expected for hydrophobic interaction with the membrane. The orientation of the peptide in DPC micelles was evaluated from the effect of incorporation of a spin-label 12-doxylstearate into the micelles. The peptide portions, found to be immersed in the micelles, include the helical segment, the bent segment, and some hydrophobic residues within the C-terminal segment. Two additional synthetic peptides, one corresponding to the NH2-terminal helical segment (TXAS residues 33-46) and the other including the bent and the C-terminal segments (TXAS residues 47-60) were analyzed for their ability to incorporate into peptidoliposomes. The helical peptide readily incorporated into liposomes; the other peptide did not. These results support the presence of a second functional membrane anchor region localized to the helical segment within TXAS residues 33-46, with passive membrane contacts in the bent and the C-terminal segments of the peptide (TXAS residues 47-60) due to immersion of the helical in the membrane.