The protein profilin binds to both actin and the head groups of poly)phosphoinositide)s and may regulate both actin assembly and the phosphoinositide signaling pathway. As a first step in understanding the activity of profilin at the molecular level, we have determined the secondary structure of Acanthamoeba profilin I in solution using multidimensional, heteronuclear NMR spectroscopy. Using a combination of triple-resonance (1H, 13C, 15N) experiments, we obtained virtually complete backbone and side-chain resonance assignments based solely on scalar couplings. 3D and 4D NOESY experiments were then used to determine the secondary structure and global fold of Acanthamoeba profilin I. The central feature of the protein structure is a five-stranded antiparallel beta-sheet flanked by three helices and a short two-stranded antiparallel beta-sheet.