Previous NMR studies have shown that many phospholipase A2 (PLA2, from bovine pancreas, overexpressed in Escherichia coli) mutants display some properties reminiscent of a molten globule state. Further NMR analyses for some of the mutants indicated that formation of the "molten globule-like state" is a pH-dependent phenomenon. The mutants I9Y and I9F showed perturbed NMR properties throughout the pH range studied, while the mutants H48A and C44A/C105A displayed native-like spectra at neutral pH but molten globule-like ones under acidic conditions, with a "transition pH" around 4. On the other hand, wild-type PLA2 exhibits exceptional pH stability and turns into a similar molten globule-like state only under highly acidic conditions such as 1 M HCl. The H48A mutant was used to rigorously establish the property of the molten globule-like state of PLA2 mutants. The results of far-UV CD, near-UV CD, and ANS-binding fluorescence suggest that H48A retains native-like secondary structures but loses tertiary structure during the conformational transition. However, the tertiary structure is not completely lost, as evidenced by the retention of some long-range NOEs in two-dimensional NOESY spectra. The conclusion was further substantiated by three-dimensional NOESY-HSQC experiments on a 15N-labeled H48A sample. It was revealed that the molten globule-like state at mildly acidic pH retained some rigid tertiary structure, which consisted of partial alpha-helix II (Y52-L58), alpha-helix III (D59-V63), beta-wing (S74-S85) and partial alpha-helix IV (A90-N97). These residual tertiary structures grouped in half of the protein could be attributed to stabilization by some of the disulfide bonds. The extreme sensitivity of the PLA2 structure to site-directed mutagenesis is unprecedented. It is interesting to note that most of the functional residues (the active site, the hydrophobic channel, the interfacial binding site, and the calcium-binding loop) are located in the remainder of the protein, which is well disrupted in tertiary interactions.