We determined the temporal and spatial localization of the phytoalexin avenanthramide A, and its biosynthetic enzyme, hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxy-cinnamoyltransferase (AsHHT) in oat leaves infected with the crown rust fungus. Accumulation of avenanthramide A and AsHHT was first observed predominantly in hypersensitive response (HR) cells at 36 h post inoculation (hpi), and later in adjacent cells at 48 hpi. At 120 hpi, avenanthramide A was detected in a wider area of infected tissues, but AsHHT-positive signals were only observed in the HR and adjacent cells, suggesting that avenanthramide A was synthesized around the HR cells, and then transported to other parts of the infected tissues in a center-to-periphery manner.