Bovine brain platelet-activating factor (PAF) acetylhydrolase isoform Ib is a heterotrimeric enzyme. Its gamma-subunit (which, formerly, we called the 29-kDa subunit) acts as a catalytic subunit, whereas the alpha-subunit (45 kDa) is the bovine homolog of the product of human LIS-1, the causative gene of Miller-Dieker lissencephaly, indicating that this intracellular PAF acetylhydrolase plays a key role in brain development. In the current study, we cloned the cDNA for the beta-subunit (30 kDa) of bovine brain PAF acetylhydrolase Ib. The predicted 229-amino acid sequence was homologous (63.2% identity) to that of the gamma-subunit, especially (86% identity) in the catalytic and PAF receptor homologous domains. The recombinant beta-protein produced in Escherichia coli showed significant PAF acetylhydrolase activity. A mutant protein, in which Ser48, which corresponds to the active serine residue of the gamma-subunit, was replaced with cysteine showed no enzymatic activity, suggesting Ser48 is the active serine residue. Although the beta- and gamma-subunits form a heterocomplex in the native enzyme, both recombinant beta- and gamma-proteins exist as a homodimer. The purified recombinant beta-protein was labeled readily with [1,3-H]diisopropyl fluorophosphate, whereas the beta-subunit in the native complex was only labeled with higher concentrations of [1,3-3H]diisopropyl fluorophosphate to a lesser extent than the gamma-subunit. Combined with our previous data, the present study demonstrated that bovine brain PAF acetylhydrolase Ib is a unique enzyme possessing two catalytic subunits and another, possibly regulatory, subunit.