We investigated nitric oxide (NO) production in Paramecium caudatum, and the role of NO production in population growth in culture and thermoregulation. Nitrite (N2(superscript -)) concentration in media containing P. caudatum [6350±390 (SD) paramecia/ml] was 2.37±0.53 μM after 6 h, compared to 0.16±0.06 μM in media alone (p<0.005), and the NO synthase (NOS) inhibitor, N(superscript ω)-nitro-L-arginine methylester (L-NAME), reduced [NO2(superscript -)] to 0.81±0.24 μM (p<0.02). Media containing P. caudatum produced [3H] L-citrulline from [3H] L-arginine, and the [3H] L-citrulline production was inhibited by L-NAME. Addition of A23187, a calcium ionophore, to the media resulted in greater [NO2(superscript -)] (1.49±0.28 μM with no A23187, 2.51±0.23 μM with 0.1 μM A23187 added, p<0.05). Western blot analysis revealed a 155 kDa protein that reacted with mouse NOS1 antibody. Paramecia concentration increased from 51±9 per ml on day 0 to 943±53 per ml on day 7. L-NAME decreased paramecia concentration at day 7 (0.1 mM, 720±70 per ml; 1.0 mM, 761±49 per ml; and 10 mM, 132±32 per ml; p<0.05 compared to control for all 3 concentrations). In a thermal gradient, P. caudatum selected an environmental temperature (Ts) of 32.9±0.3℃, addition of 10 mM L-NAME reduced Ts to 24.3±0.3℃ (p<0.05). These data suggest that P. caudatum produce NO via a calcium dependent NOS similar to mammalian NOS1, and inhibition of NO production reduced paramecia number in culture and decreased Ts.