Tricholoma matsutake is a representative mushroom species with a characteristic pleasant aroma. The characteristic aroma component is methyl cinnamate, which is also produced in many plants. In basil, cinnamic acid is produced from l-phenylalanine (l-Phe) by phenylalanine ammonia-lyase (PAL) and converted to methyl cinnamate by a cinnamate/p-coumarate carboxyl methyltransferase. Two PAL genes, Tmpal1 and Tmpal2, have been isolated from T. matsutake. In this study, we aimed to clarify the relationships between l-Phe, methyl cinnamate production, and PAL expression in the mycelium of T. matsutake strain NBRC 30605. For this purpose, methyl cinnamate content, PAL activity, and transcript levels of Tmpal1 and Tmpal2 were examined in the mycelia of T. matsutake supplemented with l-Phe. The mycelia were cultured in 20 mL of a liquid medium (2% glucose, 0.15% yeast extract, and 0.15% Bacto Soytone) at 20 °C for 45 d, supplemented with 0.5-6 mM l-Phe, and then grown for a further 15 d. Mycelia cultured without l-Phe supplementation for 60 d in the medium were used as a control. Crude extracts were prepared from the mycelia harvested for enzymatic, protein, and methyl cinnamate assays. Methyl cinnamate was measured using gas chromatography. PAL activity was assayed by measuring the rate of trans-cinnamic acid formation as the absorbance at 290 nm (ɛ290 = 10,000 M−1 cm−1). The transcript levels of Tmpal1 and Tmpal2 were examined by performing real-time reverse transcriptase-quantitative PCR on the total RNA. Methyl cinnamate was detected in very low levels in cultures without l-Phe supplementation, but its content per mg of protein increased markedly with increasing concentrations of l-Phe, especially at 4-6 mM. When 6 mM l-Phe was added to the culture medium, the methyl cinnamate content was approximately 55-fold higher than that of the control sample. The specific activity of PAL also increased in cultures supplemented with l-Phe, especially at 4-6 mM. When l-Phe was added to the culture medium, the methyl cinnamate content in the mycelia was relatively well correlated with PAL activity. These results indicated that supplementation with l-Phe, a precursor of methyl cinnamate, increases the specific activity of PAL, leading to an increase in methyl cinnamate production in the mycelia of T. matsutake. The transcript level of Tmpal1 did not change markedly with l-Phe supplementation. In contrast, the transcript level of Tmpal2 increased greatly in cultures supplemented with 4-6 mM l-Phe. These results suggested that the expression of Tmpal1 and Tmpal2 was controlled by different regulatory mechanisms and that they may have different biological functions in T. matsutake. In addition, the pattern of PAL activity in the presence of l-Phe was similar to that of the transcript level of Tmpal2, but not Tmpal1, suggesting that the increase in PAL activity was dependent on the increased transcription of Tmpal2.