The constructed strains AM63, having an extra copy of the HMG2 gene with a K6R stabilizing mutation in Hmg2p expressed under the control of the inducible galactose promoter and stably integrated into the chromosomal HO locus, and AM64, a derivative of AM63 with an additional deletion of the ERG6 gene, were used as tools to test the squalene accumulation capacity of Saccharomyces cerevisiae. Kinetic data indicated high squalene levels in the early stages of semi-anaerobic cultivation of these strains. The stable Hmg2p induced a strong increase in the squalene pool and a smaller increase in the lanosterol pool. In AM63, the squalene content was ∼20-fold higher than in the wild-type EGY48 parental strain. In AM64, the combined Hmg2p stabilization and ERG6 deletion did not further enhance squalene accumulation, as lack of ergosterol feedback inhibition led to an elevated transfer of surplus squalene into C27 sterols. The obtained maximum capacity of the selected strains to accumulate squalene and our observations provide a further understanding of the regulation of ergosterol pathway and may also be used as a reference value for its production using food-grade strains of S. cerevisiae.