Different environmental nitrogen sources play selective roles in the development of cyanobacterial blooms and noxious effects are often exacerbated when toxic cyanobacteria are dominant. Cylindrospermopsis raciborskii CS-505 (heterocystous, nitrogen fixing) and Raphidiopsis brookii D9 (non-N2 fixing) produce the nitrogenous toxins cylindrospermopsin (CYN) and paralytic shellfish toxins (PSTs), respectively. These toxin groups are biosynthesized constitutively by two independent putative gene clusters, whose flanking genes are target for nitrogen (N) regulation. It is not yet known how or if toxin biosynthetic genes are regulated, particularly by N-source dependency. Here we show that binding boxes for NtcA, the master regulator of N metabolism, are located within both gene clusters as potential regulators of toxin biosynthesis. Quantification of intra-and extracellular toxin content in cultures at early stages of growth under nitrate, ammonium, urea and N-free media showed that N-sources influence neither CYN nor PST production. However, CYN and PST profiles were altered under N-free medium resulting in a decrease in the predicted precursor toxins (doCYN and STX, respectively). Reduced STX amounts were also observed under growth in ammonium. Quantification of toxin biosynthesis and transport gene transcripts revealed a constitutive transcription under all tested N-sources. Our data support the hypothesis that PSTs and CYN are constitutive metabolites whose biosynthesis is correlated to cyanobacterial growth rather than directly to specific environmental conditions. Overall, the constant biosynthesis of toxins and expression of the putative toxin-biosynthesis genes supports the usage of qPCR probes in water quality monitoring of toxic cyanobacteria.We thank our colleagues (AWI, Bremerhaven, Germany) Annegret Muller for technical support in toxin analysis, and Bernd Krock and Urban Tillmann for critical contributions to the manuscript. Financial support for this work was provided by Grants from Chile (to Monica Vasquez): Fondecyt 1080075 and 1131037; Fondef MR10I1008; Millennium Nucleus EMBA P04/007; and "Programa de Cooperacion Cientifica Internacional (PCCI) CONICYT/DFG" between the P. Universidad Catolica de Chile and the Alfred Wegener Institute Helmholtz Centre for Polar and Marine Research.4FONDEFkstuckenm@gmail.com; Uwe.John@awi.de; Allan.Cembella@awi.de; katiasotoliebe@gmail.com; mvasquez@bio.puc.clChile; Fondecyt [1080075, 1131037]; Fondef [MR10I1008]; Millennium Nucleus EMBA [P04/007]; Universidad Catolica de Chile; Alfred Wegener Institute Helmholtz Centre for Polar and Marine ResearchFONDEF