The conserved Snf1/AMP-activated protein kinase family is one of the central components in the nutrient sensing and regulation of the carbon metabolism in eukaryotes. It is also involved in several other processes such as stress resistance, invasive growth and ageing. Snf1 kinase is composed of a catalytic alpha-subunit Snf1, a regulatory gamma-subunit Snf4 and one of three possible beta-subunits, Sip1, Sip2 or Gal83. We used a systematic approach to study the role of the three beta-subunits by analysing all seven possible combinations of beta-subunit deletions together with the reference strain. Previous studies showed that the three beta-subunits are redundant for growth on alternative carbon sources. Here we report that the mutant strain with only SIP1 expressed (sip2Delta gal83Delta) could utilize acetate, but neither ethanol nor glycerol, as alternative carbon source. We also showed that Gal83 is the most important isoform not only for the growth on non-fermentable carbon sources, but also for regulation of ergosterol biosynthetic genes, under glucose-limited condition. Furthermore, we found that Sip2, but not Sip1, can take over when Gal83 is deleted, but to a lesser extent. However, Sip1 may be sufficient for some other processes such as regulation of the nitrogen metabolism and meiosis.