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Flotation of seeds in solvents is a common means of separating unfilled and filled seeds. While a few protocols for processing red spruce (Picea rubens) seeds recommend ethanol flotation, delayed and reduced germination have been reported. We conducted an ethanol bioassay on seeds previously stored at -20°C to quantify the concentration required to separate red spruce seeds and the effects on germination. We used seeds from Canada (CAN) that had been exposed to ethanol during processing, and seeds from the United States (USA) that had not been exposed to ethanol during processing. Seeds were exposed to 10 ethanol concentrations (10-100%) and deionised water was used as a control. The effective concentration of ethanol for 50% (EC50) of the seeds to sink ranged by source from 70.9 to 90.7%, with all seeds sinking in 100% ethanol. The use of less than 100% ethanol is not adequate for seed separation, as some filled seeds could float and be mistakenly categorised as unfilled. The mean EC50 of ethanol that inhibits germination was significantly higher for USA sources (52.7%), than for CAN sources (40.8%; P < 0.05). Ethanol concentrations that inhibited germination coincided with delays in germination. The mechanism of phytotoxity was not determined; however, damage during extraction, desiccation and storage at -20°C are potential sources. We recommend separating red spruce seeds by physical means rather than ethanol flotation to avoid potential negative impacts on germination.