Cyanobacteria are capable of producing multiple microcystin variants simultaneously. The mechanisms that determine the composition of microcystin variants in cyanobacteria are still debated. [Asp3]microcystin-RR contains arginine at the position where the more toxic [Asp3]microcystin-LR incorporates leucine. We cultured the filamentous cyanobacterium Planktothrix agardhii strain 126/3 with and without external addition of leucine and arginine. Addition of leucine to the growth medium resulted in a strong increase of the [Asp3]microcystin LR/RR ratio, while addition of arginine resulted in a decrease. This demonstrates that amino acid availability plays a role in the synthesis of different microcystin variants. Environmental changes affecting cell metabolism may cause differences in the intracellular availability of leucine and arginine, which can thus affect the production of microcystin variants. Because leucine contains one nitrogen atom while arginine contains four nitrogen atoms, we hypothesized that low nitrogen availability might shift the amino acid composition in favor of leucine, which might explain seasonal increases in the [Asp3]microcystin LR/RR ratio in natural populations. However, when a continuous culture of P. agardhii was shifted from nitrogen-saturated to a nitrogen-limited mineral medium, leucine and arginine concentrations decreased, but the leucine/arginine ratio did not change. Accordingly, while the total microcystin concentration of the cells decreased, we did not observe changes in the [Asp3]microcystin LR/RR ratio in response to nitrogen limitation.