Renal medullary cells are able to accumulate glycerophosphocholine during adaptation to the high extracellular osmolality. The aim of this study was to investigate the effect of hyperosmolality on both phospholipase A2 activity and the rate of choline incorporation into glycerophosphocholine in rabbit renal medulla and Madin-Darby Canine Kidney cells. Phospholipase A2 activity was assayed in cellular subfractions isolated from both rabbit kidney medulla and Madin—Darby Canine Kidney cells in the presence of either 1-palmitoyl-2-[1-¹⁴C]palmitoyl phosphatidylcholine or 1-stearoyl-2-[1-¹⁴C]arachidonyl phosphatidylcholine as substrate. The rate of choline incorporation into glycerolphosphocholine was measured in Madin—Darby Canine Kidney cells growing in the presence of [methyl-³H]choline in the growth medium. Water deprivation of rabbits resulted in an increase of phospholipase A2 activity from 2.7 ± 0.4 (n = 5) and 5.7 ± 0.7 (n = 5) to 5.0 ± 0.8 (n = 5) and 10.8 ± 1.3 (n = 5) pmol of fatty acid released/min per mg protein in mitochondrial and microsomal fractions, respectively, using dipalmitoyl phosphatidilcholine as substrate while the activity of cytosolic enzyme remained unchanged. Similarly, the addition of sodium chloride in order to increase growth medium osmolality (from 320 mOsm/kg to 520 mOsm/kg) resulted in an elevation of both mitochondrial (from 1.8 ± 0.1 to 4.9 ± 0.8 pmol of fatty acid released/min per mg protein, (n = 4) and microsomal (from 8.7 ± 0.5 to 15.9 ± 1.7 pmol of fatty acid released/min per mg protein, n = 4) phospholipase A2 activities. An acceleration of phospholipase A2 activity in Madin—Darby Canine Kidney cells was accompanied by 1.5-fold stimulation of choline incorporation into intracellular glycerophosphocholine. In the presence of raffinose to increase the medium osmolality, phospholipase A2 was mainly activated in microsomal fraction of MDCK cells (up to 380% of the control value). The cytosolic PLA2 exhibited still low activity (1.3 ± 0.1 pmol of fatty acids released/min per mg protein) despite relatively high stimulation of this enzyme in MDCK cells growing in the raffinose containing, hyperosmotic medium. The data indicate that both animal antidiuresis in vivo as well as hyporosmolality of extracellular fluids in vitro result in an enhancement of phospholipase A2 activity in kidney medulla and renal cells in culture. It is concluded that activation of this enzyme is involved in the adaptation process of kidney to increased extracellular osmolality.