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Evidence suggests that low cobalt (Co) may limit heterocyst frequency (HF) in filamentous diazotrophic cyanobacteria. Therefore, the effect of three Co concentrations on HF in four species in the genera Aphanizomenon and Dolichospermum was examined in N‐depleted (N was not added) culture. Long‐term HF data were also analysed in experimentally fertilised N‐limited Lake 227.HF ranges and means at 0.17, 17, and 170 nmol Co/L were 4.1%–5.7% (4.6%), 5.4%–7.4% (6.4%), and 5.9%–9.3% (7.4%), respectively, after 11 days of growth in batch cultures suggesting that Co plays a role in regulating HF. HF at 0.17 nmol/L was significantly different from HF at the two higher concentrations in all species implicating Co in heterocyst differentiation. However, growth rates and final biovolume yields were not significantly affected by Co in any of the species.Applying the ratio of total phosphorus (TP)/Co in culture media (106) at 0.17 nmol Co/L to an N‐limited system implies that dissolved Co would have to be less than 1 pmol/L to severely limit HF at 1 μmol TP/L. This Co concentration is much lower than dissolved Co in Lake 227 during an Aphanizomenon skujae bloom with 1 μmol TP/L in 2017. Furthermore, Co increased from 0.7 to 2.0 nmol/L simultaneously increasing with HF and cyanobacteria biomass suggesting that Co probably did not limit HF and bloom biomass in Lake 227.Mean summer HFs in Lake 227 during 2000–2020 were 3.4% (epilimnion) and 4.0% (metalimnion), similar to HF at the lowest Co in the cultures. However, HF was significantly higher after 2015 following a shift in dominant bloom species from A. schindlerii to the smaller A. skujae. Calculations suggest that the decrease in cell size may have prompted a higher HF in A. skujae in order to fix an amount of N per unit biomass similar to that fixed by the larger A. schindlerii. Thus, two previously unidentified factors, Co and cell size, can be added to the list of factors that affect HF. [ABSTRACT FROM AUTHOR]