[35S]Methionine labeling of intact barley roots (Hordeum vulgare cv Klondike) after short (6-12 h) and longer (18-24 and 90-96 h) periods of K+ deprivation revealed that several membrane polypeptides were synthesized in significantly increased amounts following withdrawal of K+ from nutrient solutions. One of these, a 43-kD polypeptide localized in plasma membrane- and tonoplast-enriched fractions, accounted for a large part of 35S incorporation into membranes when [35S]methionine was administered for 6 h following 6 h of K+ deprivation. With increasing duration of K+ deprivation, 35S incorporation into this 43-kD polypeptide decreased. This polypeptide, referred to as KR43, was not synthesized when NO3− or inorganic phosphate was removed or when Rb+ was substituted for K+. However, it was synthesized when K+ was removed and replaced by an equivalent concentration of Na+. The intrinsic nature of this polypeptide and the time course of changes in its expression, which correspond with changes of K+(86Rb) influx associated with K+ deprivation, provide evidence that this polypeptide may form part of the high-affinity K+ transport system in barley roots. A possible role for this polypeptide is discussed in the context of changes in the subcellular distribution of K+ in barley roots following interruption of K+ supply. A 45-kD microsomal polypeptide, identified in earlier studies as a response to K+ deprivation, is suggested to be an extrinsic protein, readily displaced from membranes by exposure to ethylenediaminetetraacetate.