HI0381 of Haemophilus influenzae was investigated by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. HI0381 is a 153-residue peptidoglycan-associated outer membrane lipoprotein, and a part of the larger Tol/Pal network. Here, we report its backbone (1)H, (15)N, and (13)C resonance assignments, and secondary structure predictions. About 97% of all of the (1)HN, (15)N, (13)CO, (13)C alpha, and (13)C beta resonances covering 131 non-proline residues of the 134 residue, mature protein, were clarified by sequential and specific assignments. CSI and TALOS analyses revealed that HI0381 contains five alpha-helices and five beta-strands. To characterize the structure of HI0381, the effects of pH and salt concentration were investigated by CD. In addition, the structural changes occurring when HI0381 was in a membranous environment were investigated by comparing its HSQC spectra and CD data in buffer and in DPC micelles; the results showed that helix alpha 4 and strand beta 4 became aligned with the membrane. We conclude that the conformation of HI0381 is affected by the membrane environment, implying that its folded state is directly related to its function.