An interesting observation, reported for transgenic plants that have been engineered to overproduce osmolytes, is that they often exhibit impaired growth in the absence of stress. As growth reduction and accumulation of osmolytes both typically result from adaptation, we hypothesized that growth reduction may actually result from osmolyte accumulation. To examine this possibility more closely, intracellular proline level was manipulated by expressing mutated derivatives of tomPRO2 (a Δ1-pyrroline-5-carboxylate synthetase, P5CS, from tomato) in Saccharomyces cerevisiae. This was done in the presence and absence of a functional proline oxidase, followed by selection and screening for increased accumulation of proline in the absence of any stress. Here we show, in support of our hypothesis, that the level of proline accumulation and the amount of growth are inversely correlated in cells grown under normal osmotic conditions. In addition, the intracellular concentration of proline also resulted in increases in ploidy level, vacuolation and altered accumulation of several different transcripts related to cell division and gene expression control. Because these cellular modifications are common responses to salt stress in both yeast and plants, we propose that proline and other osmolytes may act as a signaling/regulatory molecule able to activate multiple responses that are part of the adaptation process. As in previous studies with transgenic plants that overaccumulate osmolytes, we observed some increase in relative growth of proline-overaccumulating cells in mild hyperosmotic stress.