Two new soybean [Glycine max (L.) Merr. cv. Williams] loci, designated Eu2 and Eu3, were identified in which ethyl methanesulfonate (EMS)-induced mutation eliminated urease activity. These loci showed no linkage to each other or to the "Sun-Eul" locus described in the accompanying paper (Meyer-Bothling and Polacco 1987). Unlike sun (seed urease-null) mutations those at Eu2 and Eu3 affected both urease isozymes: the embryo-specific (seed) and the ubiquitous (leaf) urease. The eu2/eu2 mutant had no leaf activity and 0.6% normal seed activity. Two mutant Eu3 alleles were recovered, eu3-e1 and Eu3-e3. The eu3-e1/eu3-e1 genotype lacked both activities while Eu3-e3/Eu3-e3 had coordinately reduced leaf (0.1%) and seed (0.1%) activities. Only the Eu3-e3 mutation showed partial dominance, yielding about 5%-10% normal activity for each urease in the heterozygous state. Each homozygous mutant contained normal levels of embryo-specific urease mRNA and protein subunit, both of normal size. However, urease polymerization was aberrant in all three mutants. In all cases where urease could be measured, it was found to be temperature sensitive and, in addition, the embryo-specific urease of Eu3-e3/Eu3-e3 had an altered pH dependence. These mutants may be defective in a urease maturation function common to both isozymes as suggested by the normal levels of urease gene product, coordinately (or nearly so) reduced urease isozyme activities, temperature sensitivity in both ureases (Eu3-e3) and the non-linkage of Eu2 and Eu3 to the locus encoding embryo-specific urease (Sun-Eul). Ubiquitous urease activity is reduced in mutant seed coat and callus culture as well as in leaf and cotyledon tissue. No mutant callus utilized urea (5 to 10 nM0 as sole nitrogen source. However, all mutant cell lines tolerated normally toxic levels of urea (25 to 250 mM) added to medium containing KNO3/NH4No3 as nitrogen source. Urea thus may be used in cell culture as a selection agent for phenotypes either lacking or regaining an active ubiquitous urease.