A procedure is described for the affinity purification of the mitotic form of anaphase-promoting complex/cyclosome (APC/C) from HeLa cells. It is based on the binding of mitotically phosphorylated APC/C to the phosphate-binding site of p13(suc1), followed by specific elution with a phosphate-containing compound. The procedure is rapid, simple, and yields 50- to 70-fold purification of soluble APC/C, with a approximately 30% recovery of activity.