Major ion chemistry data collected as part of the Environmental Protection Agency (EPA) Eastern Lake Survey was examined to evaluate the mechanisms and extent of alkalinity regulation in 37 undisturbed, softwater lakes in Florida. Comparison of major ion‐Cl ratios in atmospheric deposition and in lakewater shows the reactions resulting in retention of sulfate and nitrate are the dominant sources of alkalinity; production of organic acids and ammonium retention are the major alkalinity‐consuming processes. Based on average reactions, enrichment of major cations accounted for only 12% of net alkalinity generation in the study lakes. In general, calcium and potassium were depleted in low ANC lakes, presumably by in‐lake sinks, and were enriched in most higher ANC lakes by groundwater inputs. Differences in alkalinity among these lakes reflect hydrologic factors and the proximity of clay and carbonate deposits to the lake bed. Overall, net alkalinity generation nearly balanced H+ predicted from evaporative concentration of atmospheric acid inputs; this close balance suggests that the alkalinity status of these lakes is very sensitive to changes in atmospheric loadings and groundwater alkalinity inputs.