Abstract : The activation of phospholipase D (PLD) is a common response to mitogenic stimuli in various cell types. As PLD‐mediated signaling is known to be disrupted in the presence of ethanol, we tested whether PLD is involved in the ethanol‐induced inhibition of cell proliferation in rat cortical primary astrocytes. Readdition of fetal calf serum (FCS) to serum‐deprived astroglial cultures caused a rapid, threefold increase of PLD activity and a strong mitogenic response ; both effects were dependent on tyrosine kinases but not on protein kinase C. Ethanol (0.1‐2%) suppressed the FCS‐induced, PLD‐mediated formation of phosphatidic acid (PA) as well as astroglial cell proliferation in a concentration‐dependent manner. Moreover, exogenous bacterial PLD increased astroglial proliferation in an ethanol‐sensitive manner, whereas exogenous PA or lysophosphatidic acid was less effective. Formation of PA and astroglial proliferation were strongly inhibited by 1‐butanol (0.1‐1%), a substrate of PLD, but were unaffected by t‐butanol, a nonsubstrate ; 2‐butanol had intermediate effects. Platelet‐derived growth factor and endothelin‐1 mimicked the mitogenic effect of FCS ; their effects were also inhibited by the butanols in the potency order 1‐butanol > 2‐butanol > tert‐butanol. Our results, in particular, the differential effects of 1‐, 2‐, and tert‐butanol with respect to PA formation and astroglial proliferation, strongly suggest that the antiproliferative effects of ethanol in glial cells are due to the disruption of the PLD signaling pathway. This mechanism may also contribute to the inhibition of astroglial growth and brain development observed in alcoholic embryopathy.