A reverse-phase HPLC protocol for analysis of strictly hydrophobic peptides and proteins was developed. Peptide aggregation is minimized by using only 25-40% water in methanol or ethanol as initial solvents and subsequent elution with a gradient of propan-2-ol. Analysis of the pulmonary surfactant-associated proteins B (SP-B) and C (SP-C) with this method reveals several features. (1) SP-B and SP-C retain their secondary structures and separate by about 15 min over a 40 min gradient. SP-B is more hydrophilic than SP-C, which in turn behaves chromatographically like palmitoyl-ethyl ester. (2) SP-C exhibits isoforms additional to the major form characterized previously, which contains two thioester-linked palmitoyl groups. The isoforms now observed contain one or three palmitoyl moieties and constitute together 15-20% of the major form. The tripalmitoylated species contains a palmitoyl group linked to the epsilon-amino group of Lys-11, as concluded from the elution position,MS and amino acid sequence analysis. The tripalmitoylated form increases relative to the dipalmitoylated form on incubation of SP-C ina phospholipid environment. An Nepsilon-bound palmitoyl moiety constitutes a third mode of fatty acyl modification of proteins, in addition to the established Nalpha-bound myristoyl groups and S-bound palmitoyl chains. (3) The dimeric structure of SP-B, lacking covalent modifications, is confirmed by MS detection of the dimer. No SP-B isoforms were detected. (4) Denatured, non-helical SP-C can be distinguished chromatographically from the native alpha-helical peptide. (5) HPLC of SP-C at 60-75 degrees C reveals an isoform containing an extra 14 Da moiety compared with the main form. This is concluded to arise from inadvertent methyl esterification of the C-terminal carboxy group. In conclusion, this HPLC method affords a sensitive means of assessing modifications and conformations of SP-B or SP-C in different disease states and before functional studies. It might also prove useful for analysis of other strictly hydrophobic polypeptides.