The accumulation and transport of solutes are hallmarks of osmoadaptation. In this study we have employed the inability of the Saccharomyces cerevisiae gpd1 gpd2 mutant both to produce glycerol and to adapt to high osmolarity in order to study solute transport through aquaglyceroporins and the control of osmostress-induced signaling. High levels of different polyols, including glycerol, inhibited growth of the gpd1 gpd2 mutant. This growth inhibition was suppressed by expression of the hyperactive allele Fps1-1 of the osmogated yeast aquaglyceroporin, Fps1. The degree of suppression correlated with the relative rate of transport of the different polyols tested. Transport studies in secretory vesicles confirmed that Fps1-1 transports polyols at increased rates compared with wild type Fps1. Importantly, wild type Fps1 and Fps1-1 showed similarly low permeability for water. The growth defect on polyols in the gpd1 gpd2 mutant was also suppressed by expression of a heterologous aquaglyceroporin, rat AQP9. We surmised that this suppression was due to polyol influx causing the cells to passively adapt to the stress. Indeed, when aquaglyceroporin-expressing gpd1 gpd2 mutants were treated with glycerol, xylitol or sorbitol the osmosensing HOG pathway was activated and the period of activation correlated with the apparent rate of polyol uptake. This observation supports the notion that deactivation of the HOG pathway is closely coupled to osmotic adaptation. Taken together, our XconditionalX osmotic stress system facilitates studies on aquaglyceroporin function and reveals features of the osmosensing and signaling system.