Plants of the hardy cultivar of alfalfa, Rambler (Medicago media Pers.) were hardened either at 1–2 °C or at 20/17 °C (day/night) at various soil moistures to determine whether cold hardening has a direct influence on the accumulation of free proline and total sugars in plant tissues or if the accumulation is due to a drought effect caused by the cold hardening temperatures. The very low soil moisture (25% of the water holding capacity) caused a dry state in the plants, increased the cold tolerance (LT50), the proline content and the percentage of the total sugars and dry matter of the tissues. High soil moisture (saturated and flooded soils) generally had opposite effects. The cold hardening of plants at 1–2 °C in the very low soil moisture for 2 or 3 weeks caused a lower accumulation in the proline content of leaves and crowns than the hardening at 20/17 °C. However, in these conditions, the total sugars and the LT50 in cold-hardened plants increased at a higher pace than in plants hardened at 20/17 °C. These results favor the hypothesis that the proline accumulation in cold-stressed plants is not directly caused by the cold hardening temperatures while sugars are, but is due to a drought effect related to the cold hardening.Key words: Proline, sugars, alfalfa, soil moisture, cold tolerance, drought stress