Opine is the generic name of the strain-specific metabolites in crown gall and hairy root induced by Agrobacterium tumefaciens and A. rhizogenes, respectively. While various opines are known from crown gall, only two opines were hitherto known in hairy root. Kamada et al. found a new opine, termed mikimopine (1), in hairy root of tobacco induced by A. rhizogenes NIAES1724 via high-voltage paper electrophoresis. In this paper, we describe the isolation and structural elucidation of mikimopine (1) and its lactam (2). Hairy root of tobacco was extracted with water, purified by charcoal and Sephadex LH20 column chromatography to yield 1. Mikimopine (1) was acid-labile and converted to lactam (2) with 0.5N HCl. Structural analysis using FABMS, ¹H-NMR, ¹³C-NMR, 2D-NMR (including HMBC), and chemical synthesis (condensation of L-histidine and α-ketoglutaric acid) revealed the structures of 1 and 2. The molecular formula of 2 was C₁₁H₁₁N₃O₅, containing a 4,5-disubstituted imidazole ring and lactam. Biogenetically, 1 and 2 are proposed to derive from histidine and α-ketoglutaric acid via a Pictet-Spengler-like reaction. Comparison with cucumopine (reported by Davioud et al.) showed cucumopine is a diastereomer of 1, likely the synthetic epimer (4) of 1. Thus, the structure of mikimopine was concluded to be 1, and its derivative (2) is mikimopine lactam.