ABSTRACT Mutations were induced in two clonal lines of pennyroyal (Mentha pulegium L.) differing in response to the proline analog L-azetidine-2-carboxylic acid (A2C), using γ-rays and ethyl methane sulfonate (EMS). Mutants with increased biomass, an increased essential oil concentration, and increased levels of pulegone within the essential oil, compared with unmutated controls, were successfully developed in the clonal line susceptible to A2C using both the γ-rays and the EMS. Positive mutation responses were observed in almost all the mutants. A mutant developed using γ-ray treatment had a 16 percent increase in dry weight, a 9 percent increase in essential oil, and a 43 percent increase in pulegone compared with an unmutated control. A mutant developed using EMS appeared to be the best overall mutant with a 32 percent increase in dry weight, a 17 percent increase in essential oil, and a 47 percent increase in pulegone.