Mutations in the gene encoding for 4-hydroxy-2-oxoglutarate aldolase (HOGA) are associated with an excessive production of oxalate in Primary Hyperoxaluria type 3 (PH3). This enzyme is the final step of the hydroxyproline degradation pathway within the mitochondria and catalyzes the cleavage of 4-hydroxy-2-oxoglutarate (HOG) to pyruvate and glyoxylate. No analyses have been performed to assess the consequences of the mutations identified, particularly for those variants that produce either full-length or nearly full-length proteins. In this study, the expression, stability, and activity of nine PH3 human HOGA variants were examined. Using recombinant protein produced in Escherichia coli as well as transfected Chinese hamster ovary (CHO) cells, it was found that all nine PH3 variants are quite unstable, have a tendency to aggregate, and retain no measurable activity. A buildup of HOG was confirmed in the urine, sera and liver samples from PH3 patients. To determine how HOG is cleaved in the absence of HOGA activity, the ability of N-acetylneuraminate aldolase (NAL) to cleave HOG was evaluated. NAL showed minimal activity towards HOG. Whether the expected buildup of HOG in mitochondria could inhibit glyoxylate reductase (GR), the enzyme mutated in PH2, was also evaluated. GR was inhibited by HOG but not by 2-hydroxyglutarate or 2-oxoglutarate. Thus, one hypothetical component of the molecular basis for the excessive oxalate production in PH3 appears to be the inhibition of GR by HOG, resulting in a phenotype similar to PH2.