Phosphate binds to the reduced, catalytically active form of pig allantoic fluid acid phosphatase (λmax = 510 nm) at pH 4.9, causing a very rapid spectral shift to a λmax of 540 nm. This spectral shift has permitted the determination of a Kd value of ∼6mM for phosphate binding to the reduced enzyme. Phosphate also greatly potentiates the conversion of the reduced form of the enzyme to a violet, catalytically inactive form. [32P] Phosphate was used to show that phosphate is bound very tightly in this violet form of the enzyme, in a 1:1 complex. The tightly bound phosphate may be removed by reduction of the enzyme or by treatment with 6 M guanidinium chloride. Some previous studies on iron-containing acid phosphatases may well have been complicated by the presence of variable amounts of tightly bound phosphate. © 1982 Academic Press, Inc.