The alterations in the immune system caused by ethanol appear to be a complex combination of direct and indirect effects. The role of ethanol as an osmolyte has previously been studied in this laboratory with rat splenocytes. In the present study the osmotic effects of ethanol were investigated in lymphocytes from human normal subjects and alcohol abusers. Mitogen-stimulated lymphocytes were cultured in vitro with ethanol in hyperosmotic isotonic or iso-osmotic hypotonic conditions. The former conditions mimic the physiological situation where ethanol increases osmolality in an electrolyte-balanced environment. Under these conditions, lymphocyte proliferation was unaffected. Ethanol addition in iso-osmotic hypotonic conditions, where there is electrolyte imbalance, was associated with inhibition of T-lymphocyte proliferation. Hyperosmotic hypertonic solutions in the absence of ethanol also resulted in inhibition of T-lymphocyte proliferation. Electron microscopy and measurement of cell viability and metabolic activity (lactate and ATP levels) indicated that the decreased proliferation associated with NaCl-induced hyperosmotic hypertonic conditions was at least partially attributable to cell death together with, and possibly caused by, detrimental effects on mitochondria. Conversely, decreased T-lymphocyte proliferation in iso-osmotic hypotonic high ethanol solutions, appeared not to be due to changes in cell viability, nor alterations to energy metabolism. It is proposed that ion fluxes involved in the maintenance of cell volume, in particular K ⁺ movement, may be important in facilitating normal lymphocyte proliferation in the presence of ethanol in pathological conditions associated with electrolyte imbalance.