Counter-selections for the loss of introduced plasmid sequences are useful for gene manipulations in yeast. We have used GAL10 promoter-mediated overexpression of GIN sequences, which inhibit the growth of cells, to develop a novel counter-selection system. Yeast cells carrying a GIN sequence grow normally on glucose medium but are unable to grow on galactose medium, whereas derivatives that have lost the GIN sequence are able to grow in the presence of galactose. We constructed autonomously replicating, integrating, and disruption plasmids carrying GIN sequences and tested their use to select for loss of the plasmid. The results showed that the GIN sequences provide a selection for efficient loss of plasmids or integrated constructs from yeast during growth on galactose medium, indicating that this system can be used for plasmid shuffling, gene replacements and marker gene recycling. This counter-selection system has wide application, because any Gal+ strain and a wide variety of marker genes can be used. In addition, counter-selection systems using growth-inhibitory sequences should be applicable to other yeasts and possibly to other organisms.