1. Impacts of three cobalt (Co) concentrations were examined on heterocyst frequency and growth rate in four diazotrophic cyanobacteria species in nitrogen (N)-depleted culture and growth rate in one non-diazotrophic species in N-replete culture. After 11 days in batch culture, heterocyst frequency (HF, % of all cells that are heterocysts) increased from 4.1-5.7% to 5.4-7.4% to 5.9-9.3% at 0.17, 17 and 170 nmol L -1 Co, implicating Co in heterocyst differentiation. Growth rate was not significantly affected by Co in any of the species suggesting that the impact of low Co on other metabolic pathways was minimized.2. Stoichiometric extrapolation of culture results to N-limited natural systems with lower nutrient concentrations infers that HF could be limited by sub-nanomolar Co concentrations.3. In experimentally fertilized N-limited Lake 227, mean summer HF in 2000-2020 was 3.4% (epilimnion) and 4.0% (metalimnion). However, in 2017 (the only year for which Co data are available) dissolved Co increased from 0.7 to 2.0 nmol L -1 during the bloom simultaneously with increasing HF and cyanobacteria biomass, hence, Co probably did not limit HF and biomass. HF was significantly higher after 2015 following a shift in dominant bloom species from Aphanizomenon schindlerii to smaller A. skujae. The smaller cell size may have required a higher HF in order to maintain a relatively constant supply rate of fixed N per unit biomass.4. Surveys of ambient Co in over 280 aquatic systems across Canada and elsewhere indicate that Co is sometimes low enough to theoretically limit HF in N-limited waters. However, numerous variables influence HF so a clear understanding of relationships between Co and HF in natural systems remains elusive.