Quantitative determination of the biological activity of botulinum toxin type A usually depends on the LD(50) method after intraperitoneal injection into mice. This method requires a large number of mice to determine the toxic activity at a high level of precision and 3-4 days to obtain the results. Techniques to replace the LD(50) method have been attempted at various institutes. As a substitute for this method, by directly measuring the inhibition of neuromuscular transmission after the administration of a toxin, a method to quantitatively assess the toxin's activity by determining the compound muscle action potential (CMAP) was examined. Toxin solutions were injected into the rat gastrocnemius muscle, and that of the CMAP amplitude was determined over time. The CMAP amplitude decreased over 4 days after the injection of the toxin, and then slowly recovered. A dose-response relationship was noted for each dose, and a linear relation was observed between 0.01 and 30 U on the 1st day. From these results, we propose the CMAP as a substitute for the LD(50) method to examine the activity of toxin products as it is simple and reliable, reduces the number of experimental animals required, and lowers pain levels.