Uteroglobin (UG) is an anti-inflammatory, secreted protein with soluble phospholipase A, (sPLA,)-inhibitory activity. However, the mechanism by which UG inhibits sPLA, activity is unknown. UG is a homodimer in which each of the 70-amino acid subunits forms four alpha-helices. We previously reported that sPLA(2)-inhibitory activity of UG may reside in a segment of alpha-helix 3 that is exposed to the solvent. In addition. it has been suggested that UG may inhibit sPLA(2) activity by binding and sequestering Ca-`(++) essential for sPLA(2) activation. By site-specific mutation, we demonstrate here that Lys 43 Glu Asp 46 Lys or a combination of the two mutations in the full-length, recombinant human UG (rhUG) abrogates its sPLA(2)-inhibitory activity. We demonstrate further that recombinant UG does not bind Ca++ although when it is expressed with histidine-tag (H-tag) it is capable of binding Ca+-. Taken together our results show that: (i) Lys 43 and Asp 46 in rhUG are critical residues for the sPLA(2)-inhibitory activity of UG and (ii) Ca++-sequestration by rhUG is not likely to be one of the mechanisms responsible for its sPLA(2)-inhibitory activity. (C) 2002 Elsevier Science (USA). All rights reserved.