Mullerian inhibiting substance (MIS), a large glycoprotein secreted by the fetal and neonatal testis, is responsible for regression of the Mullerian ducts in the male embryo. This fetal growth regulator has been purified more than 2000-fold from crude testicular incubation medium following fractionation on a triazinyl dye affinity support. A high yield of 60% recovered activity was achieved in the absence of exogenous carrier protein by stabilizing MIS with 2-mercaptoethanol, EDTA, and Nonidet-P40 and eliminating losses in the handling and concentration of MIS fractions. Although affinity elution with nucleotides has proved successful in other systems, MIS could not be eluted with ATP, GTP, or AMP, with or without divalent metal ions. Nucleotide elution, however, does remove contaminating proteins prior to MIS recovery with high ionic strength. The 2000-fold-purified MIS fraction, although not homogeneous, shows a reduction-sensitive band after SDS-gel electrophoresis that has been proposed to be the MIS dimer.