Calcium-dependent phosphatidylinositol-specific phospholipase C from Streptomyces antibioticus (saPLC1) catalyzes the cleavage of phosphatidylinositol (PI) by an unusual mechanism involving a 1,6-cyclization with formation of inositol trans-1,6-cyclic phosphate (1,6-IcP), rather then inositol cis-1,2-cyclic phosphate (11,2-IcP). This conclusion has been reached based on the comparison of the released cyclic phosphate intermediate by the H16A mutant of saPLC1 with a genuine 1,6-IcP synthesized by a chemoenzymatic approach.