The content of 18 free amino acids in 34 Spanish artisanal cheeses was measured. The amino acids were determined by high performance liquid chromatography (HPLC) with pre-column derivatization, reversed-phase separation, and UV detection. The total content of free amino acids in the cheese samples ranged from 0.19 g/kg to 67.95 g/kg. The amount of lactic acid bacteria (LAB), the main microflora in cheese, is also reported. Considerable amounts of free amino acids, such as gamma-aminobutyric acid (GABA; average 330 ± 50 mg/kg) and ornithine (Orn; 450 ± 60 mg/kg), were found in some samples, thus indicating specific microbial activities. GABA concentrations were not influenced by the kind of ripening or milk origin (p