K+ and Cl- transport using 42K+ and 36Cl- was studied in primary astrocyte cultures prepared from neonatal rat brains. A component of 42K+ uptake was sensitive to both furosemide and bumetanide with maximum inhibition being obtained at 1 and 0.01 mM concentrations of the inhibitors, respectively. Furosemide and bumetanide also markedly inhibited uptake of 36Cl-. 42K+ uptake in the presence of ouabain was also sensitive to the omission of medium Na+ and Cl-. These results suggest the existence of a K+ + Na+ + Cl- cotransport system in astrocyte cultures which in many cells has been shown to be involved in volume regulation. We studied volume changes using uptake of [14C]3-O- methyl-D-glucose ([14C]3-OMG), and also ion transport, in attached cells in response to exposure to hyper- or hypotonic medium. Exposure to medium made hypertonic with mannitol resulted in shrinkage of the [14C]3-OMG space of the cells, but did not affect 36Cl- content, expressed as nmol/mg protein. Exposure to hypotonic medium led to a marked increase in the [14C]3-OMG space, rapidly followed by a decrease towards control values. After the cells were then exposed to isotonic medium there was an immediate decrease followed by a slower increase in the [14C]3-OMG space. The increase in the [14C]3-OMG space was partially inhibited by 1 mM furosemide.(ABSTRACT TRUNCATED AT 250 WORDS)