LAN analyses appear to have diagnostic value in fish pathobiology and studies were undertaken to determine optima for substrate concentration, pH, reaction time, temperature, and buffer ions. Citrate ion did not inhibit LAN at anticoagulant levels, but cyanide, pyrophosphate, and EDTA had an inhibitory effect. Storage of samples at —10 and 1 C resulted in small but significant reductions of LAN activity, while at room temperature enzyme activity was rapidly lost. LAN activity was distributed among liver fractions as follows: microsomes, 12%; mitochondria, 9%; cellular sap, 37%; other, 50%. Three isozymes of LAN were found. Blood plasma contained significant amounts of LAN activity which was significantly higher in cold- than in warm-acclimated fish. However, these LAN levels were comparable when their activity was extrapolated to body temperatures.