Reactivity with α-ketobutyrate and the effect of heat inactivation were used to reassess the relative merits of HBD and LDH as diagnostic aids in liver and heart disease. Purified LD1 and LD5 both reacted with α-ketobutyrate over a wide range of temperature and substrate concentrations. No one set of conditions adequately discriminated between these isoenzymes. Electrophoretic studies demonstrated that heat inactivation at 60° resulted in minimal loss of cardiac isoenzyme (LD1–2) activity after 1 h, while eliminating LD3–5 activities within 30 min. Estimation of the percentage of the total LDH activity remaining after heat treatment revealed a clear demarcation between patients with liver and heart disease, whilst poorer discrimination was observed with HBD. The value of pre- and post-heat LDH estimations in the differential diagnosis of liver and heart disease was demonstrated in patients with minimally elevated liver and cardiac enzymes.