The arsenate poisoning of R17 phage eclipse in Escherichia coli cultures grown in glycerol-containing medium has been found to be mediated by a dramatic loss in cell-associated F pili. Poisoning was very rapid and was nearly complete within 3 min at 37 C. The loss of pili was reflected by a 90% reduction in the ability of these cells to attach ribonucleic acid phage and by a reduction in the pili-per-cell ratio from 1.36 to 0.04 as determined by electron microscopy. Neither the integrity of the pilus per se nor the attachment process was affected by arsenate, for cell-free pili treated with arsenate retained the ability to attach phage. The arsenate effect on F piliation could be reversed readily without any loss in cell viability. This restoration of pili occurred under conditions of inhibited protein synthesis, implying that pools of pili protein exist in cells. In contrast to this phenomenon, cells grown in glucose-containing media were mainly resistant to arsenate poisoning as determined by phage attachment and the number of pili per cell. These results implied that arsenate poisons the ability of cells to synthesize and maintain F pili under certain specific conditions. The possible mechanism of this poisoning is discussed.