When a non-ionic surfactant, Span 20, was added to a mated culture of Blakeslea trispora, beta-carotene production increased 15 fold compared to that without the surfactant, reaching 2.3 g/l. The marked increase of beta-carotene was closely related to the titer of trisporic acid, a strong stimulator of beta-carotene synthesis. With Span 20, the microorganism produced 15 times more trisporic acid (8.8 mg/g dry cell weight) than without the surfactant (0.6 mg/g dry cell weight). Therefore, it was concluded that Span 20 considerably enhanced the production of trisporic acid, which subsequently stimulated beta-carotene synthesis.