Mice carrying the [Gus]H haplotype of the beta-glucuronidase gene complex have considerably decreased enzyme levels and a decreased rate of enzyme synthesis. This is now shown to result from the action of two regulatory loci within the gene complex. One is a systemic regulator, Gus-u, that acts cis to cause a uniform reduction in enzyme levels in all tissues. The other is a temporal locus, Gus-t, that acts trans to cause abrupt switches in the rate of enzyme synthesis in only certain tissues and at characteristic stages of development. The distinction between these two loci was made possible by the introduction of a method for quantitating the relative numbers of A and H allozyme subunits in beta-glucuronidase tetramers. The procedure involves purification of the enzyme, cleavage at methionyl residues with CNBr, isoelectric focusing to separate the peptides, and quantitation of the peptide containing the A/H amino acid substitution. The presence of a trans-acting regulatory locus within a gene complex raises evolutionary and functional questions about why it is located there and how it acts.