Aims: To determine the amino acid requirement and utilization pattern of Corynebacterium diphtheriae during growth and toxin production. Methods and Results: Comparing across different batches of beef-based media, the growth and toxin yield were correlated significantly with nine of the amino acids. The amino acid utilization pattern during growth of C. diphtheriae further showed that only four of the nine amino acids, namely cystine, histidine, aspartate and methionine, were critical for growth of the vaccine strain. Further investigations using synthetic media with combinations of amino acid supplements demonstrated that among the four, cystine was the most growth limiting. Conclusions: Only certain amino acids are critical for growth and toxin production by C. diphtheriae, cystine being the single most important. Significance and Impact of the Study: Owing to the potential threat from Bovine Spongiform, Encephalopathy (BSE), a need is recognized by vaccine manufacturers to substitute beef-based production media. An understanding of the specific amino acid requirements would help to develop and optimize alternative production media.