The mechanism of volume regulation in hypotonic media was analysed in human peripheral blood mononuclear (PBM) cells. Electronic cell sizing showed that hypotonic swelling is followed by a regulatory volume decrease (RVD) phase. This was confirmed by both electron microscopy and by cellular water determinations. The rate of regulatory shrinking was proportional to the degree of hypotonicity in the 0.5-0.9 X isotonic range. Cell viability was only marginally affected in this range. The content of cellular K+ decreased during RVD, while Na+ content remained unchanged. Similarly, the efflux of 86Rb (used as a K+ analog) increased upon dilution, whereas 22Na efflux was not altered. 86Rb uptake was enhanced by hypotonic stress and both ouabain-sensitive and -insensitive components were affected. A ouabain-sensitive stimulation was also seen in Na+- free media. Ouabain partially inhibited RVD only if added to the cells hours before hypotonic challenge. A normal shrinking response was observed in K+-free media, and also in Na+-free media when Li+, choline+, or Tris+ were the substitutes. In high K+ or RB+ hypotonic media shrinking was absent and a second swelling phase was observed. Cs+ displayed an intermediate behavior, with shrinking observed at lower dilutions and secondary swelling at higher ones. The direction and magnitude of the response also changed when the external K+ concentration was varied and, with 50 mM K+, no regulatory volume change occurred following hypotonic stress. These findings suggest that RVD occurs largely by a passive loss of cellular K+, resulting from a selective increase in permeability to this ion. In addition, the (Na-K) pump appears to be activated upon cell swelling by a mechanism other than Na+ entry into the cell, but this activation is not essential for RVD.