The effect of subacute administration to rats of di-isopropyl phosphorofluoridate (DFP) on the sensitivity of the cardiovascular system to carbachol and on the sensitivity of various isolated tissues to acetylcholine, methacholine and carbachol was investigated. Cholinesterase inhibition produced by DFP in the isolated tissues was also determined. In the cardiovascular experiments both the magnitude of responses and the slope of the dose-response curves for hypotension and bradycardia produced by carbachol in the anaesthetized rat were reduced in the DFP-treated group. In experiments with isolated tissuesfrom DFP-treated animals it was found that sensitivity to methacholine and acetylcholine increased in the bladder to a greater extent that in the atria or ileum. The sensitivity to carbachol decreased to a similar extent in all 3 tissues. Acetylcholinesterase and butyrylcholinesterase levels in the bladder were inhibited to a greater extent than in the other 2 tissues. The results show that the degree of enzyme inhibition produced by DFP in the tissues after subacute administration for 10 days does not correlate with the decrease in the sensitivity of the tissues to carbachol. It is suggested that the association between accumulation of transmitter and enzyme inhibition is a more important factor than the degree of enzyme inhibition itself in the development of subsensitivity to carbachol. The release of cholinergic transmitter from the electrically stimulated isolated rat bladder may also be impaired following subacute administration of DFP.