ABSTRACT Cylindrospermopsin (CYN) and 7-deoxy-cylindrospermopsin (dCYN) are potent hepatotoxic alkaloids produced by numerous species of cyanobacteria, including the freshwater Cylindrospermopsis raciborskii . C. raciborskii is an invasive cyanobacterium, and the study of how environmental parameters drive CYN production has received significant interest from water managers and health authorities. Light and CO ₂ affect cell growth and physiology in photoautotrophs, and these are potential regulators of cyanotoxin biosynthesis. In this study, we investigated how light and CO ₂ affect CYN and dCYN pool size as well as the expression of the key genes, cyrA and cyrK , involved in CYN biosynthesis in a toxic C. raciborskii strain. For cells growing at different light intensities (10 and 100 μmol photons m ⁻² s ⁻¹ ), we observed that the rate of CYN pool size production (μ _CYN ) was coupled to the cell division rate (μ _c ) during batch culture. This indicated that CYN pool size under our experimental conditions is constant and cell quotas of CYN (Q _CYN ) and dCYN (Q _dCYN ) are fixed. Moreover, a lack of correlation between expression of cyrA and total CYN cell quotas (Q _CYNs ) suggests that the CYN biosynthesis is regulated posttranscriptionally. Under elevated CO ₂ (1,300 ppm), we observed minor effects on Q _CYN and no effects on expression of cyrA and cyrK . We conclude that the CYN pool size is constitutive and not affected by light and CO ₂ conditions. Thus, C. raciborskii bloom toxicity is determined by the absolute abundance of C. raciborskii cells within the water column and the relative abundance of toxic and nontoxic strains.