The marine non-heterocystous cyanobacterium Trichodesmium fixes atmospheric N2 aerobically in light. In situ immunolocalization/light microscopy of NifH revealed that lighter, non-granulated cell regions observed correspond to the nitrogenase-containing diazocyte clusters in Trichodesmium IMS101. The number of diazocyte clusters per trichome varied from 0 to 4 depending on trichome length. The constant percentage of diazocytes (approx. 15 %) in cultured strains and five natural populations suggests a developmentally regulated differentiation process. Real-time RT-PCR showed that ntcA, encoding the global nitrogen regulator in cyanobacteria, and hetR, the key regulatory gene in heterocyst differentiation, are both constitutively expressed during a 12 h/12 h light/dark cycle. hetR in addition showed a distinct peak in the dark (close to midnight) while nifH expression commenced 6–8 h later. The expression of all three genes was negatively affected by addition of ammonia. Some early heterocyst differentiation genes were also identified in the genome of Trichodesmium. The data suggest that hetR and ntcA may be required for development and function of diazocytes in Trichodesmium.