Amino acid requirements of a Chlamydia trachomatis were defined in McCoy cell monolayer cultures, depleted of their nutrient reserves by maintenance in Earle balanced salt solution for at least 2 days, and then treated at infection with 1 microgram of cycloheximide per ml, an inhibitor of host cell protein and deoxyribonucleic acid synthesis. Serum-free Waymouth MB 752/1 medium lacking one amino acid or a group of related amino acids was added to depleted cultures before infection. Eight of the amino acids normally present in MB 752/1 were essential for chlamydial growth, six were advantageous for growth, and five were nonessential.