Two forms of ATP sulfurylase have been purified to homogeneity from mitochondria (ATPSm) and cells (ATPSc) of Euglena gracilis Klebs var. bacillaris Cori (aplastidic mutant W10BSmL). Both forms are monomeric, ATPSc is 52.3 kDa and ATPSm is 55 kDa. The pI is 7.9 for ATPSc and 5.8 for ATPSm. Therefore, ATPSm binds to DEAE-cellulose at pH 7.4; ATPSc does not. After cleavage by CNBr, the two forms of ATP sulfurylase show different sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns, suggesting that they differ in amino acid sequence. ATPSm is mainly associated with the mitochondrial membrane and ATPSc is mainly soluble in the cells. Both enzymes require similar conditions in the molybdolysis assay, but show different pH optima when sulfate is used as substrate. ATPSc is more sensitive to adenosine 5'-phosphosulfate (APS) inhibition than ATPSm in the SO2-4 incorporation reaction. In the reverse reaction, ATPSc requires much higher concentrations of PPi and MgCl2 to saturate the reaction than ATPSm. The data indicate that the two enzymes are quite distinct and may have different roles in cell metabolism.