We previously reported that the forest environment enhanced human natural killer (NK) cell activity, the number of NK cells, and intracellular anti-cancer proteins in lymphocytes, and that the increased NK activity lasted for more than 7 days after trips to forests both in male and female subjects. To explore the factors in the forest environment that activated human NK cells, in the present study we investigate the effect of essential oils from trees on human immune function in twelve healthy male subjects, age 37-60 years, who stayed at an urban hotel for 3 nights from 7.00p.m. to 8.00a.m. Aromatic volatile substances (phytoncides) were produced by vaporizing Chamaecyparis obtusa (hinoki cypress) stem oil with a humidifier in the hotel room during the night stay. Blood samples were taken on the last day and urine samples were analysed every day during the stay. NK activity, the percentages of NK and T cells, and granulysin, perforin, granzyme AlB-expressing lymphocytes in blood, and the concentrations of adrenaline and noradrenaline in urine were measured. Similar control measurements were made before the stay on a normal working day. The concentrations of phytoncides in the hotel room air were measured. Phytoncide exposure significantly increased NK activity and the percentages of NK, perforin, granulysin, and granzyme AlB-expressing cells, and significantly decreased the percentage of T cells, and the concentrations of adrenaline and noradrenaline in urine. Phytoncides, such as a-pinene and -pinene, were detected in the hotel room air. These findings indicate that phytoncide exposure and decreased stress hormone levels may partially contribute to increased NK activity.The forest environment has been enjoyed by humans for a long time because of the quiet atmosphere, beautiful scenery, mild climate, and clean fresh air. We previously reported that the forest environment enhanced human natural killer (NK) cell activity, the number of NK and NKT cells, and intracellular anti-cancer proteins in lymphocytes, and that the increased NK activity lasted for more than 7 days after the trips to forests both in male and female subjects (1-5). However, it is not clear what kind of factors in the forest environment activated human NK cells. We speculate that aromatic volatile substances derived from trees, including monoterpenes and sesquiterpenes, called phytoncides, such as a-pinene and limonene (6), may play an important role. Thus, the effects of phytoncides, such as a-pinene,