Various factors affecting N2 fixation of a cultured strain of Trichodesmium sp. (GBRTRLI101) from the Great Barrier Reef Lagoon were investigated. The diurnal pattern of N2 fixation demonstrated that it was primarily light-induced although fixation continued to occur for at least 1 h in the dark in samples that had been actively fixing N2. N2 fixation was dependent on the light intensity and stimulated more by white light when compared with blue, green, yellow and red light whereas rates of N2 fixation decreased most under red light. Inorganic phosphorous concentrations in the lower range of treatments up to 1.2 μM significantly stimulated N2 fixation and further additions promoted little or no increase in N2 fixation. Organic phosphorous (Na-glycerophosphate) also stimulated N2 fixation rates. Added combined nitrogen (NH4+, NO3−, urea) of 10 μM did not inhibit N2 fixation in short-term studies (first generation), however it was depressed in the long-term studies (fifth generation).