Mitogen-activated protein (MAP) kinases are activated by osmotic stress in a variety of cells, but their function and regulation in renal tubules is poorly understood. The present study was designed to examine the osmotic regulation of MAP kinases in the medullary thick ascending limb (MTAL) of the rat and to determine their possible role in the hyperosmotic inhibition of absorption in this segment. Tissues from the inner stripe of the outer medulla and microdissected MTALs were incubated at 37°C in control (290 mosmol/kgH₂O) or hyperosmotic (300 mM added mannitol) solution for 15 min. Activities of extracellular signal-regulated kinase (ERK), c-Jun NH₂-terminal kinase (JNK), and p38 MAP kinase were then measured using immune complex assays. Hyperosmolality increased p38 MAP kinase activity (2.3-fold) and ERK activity (2.0-fold) but had no effect on JNK activity (1.1-fold). Exposure to hyperosmolality for various times showed that the activation of p38 MAP kinase was rapid (≤5 min) and was sustained for up to 60 min, whereas the activation of ERK was transient (ERK activity peaked at 15 min, then declined to basal levels at 30 min). Pretreatment with the MAP kinase kinase inhibitor PD98059 (15 μM) blocked the hyperosmotic activation of p38 MAP kinase and ERK but did not prevent hyperosmotic inhibition of absorption. These results show that hyperosmolality differentially activates p38 MAP kinase and ERK in the MTAL. In contrast, we found no evidence for involvement of JNK in the early response to hyperosmotic stress. Eliminating the activation of p38 MAP kinase and ERK does not prevent hyperosmotic inhibition of absorption, suggesting that hyperosmolality inhibits apical membrane Na⁺/H⁺exchange (NHE3) activity via a signaling pathway distinct from these MAP kinase pathways.