1. Substance P (SP) was measured in acid acetone extracted plasma of cats using a sensitive radioimmunoassay. The immunoreactive material was submitted to ion exchange chromatography and at least 90% of immunoreactivity co-chromatographed with synthetic SP. 2. The level of immunoreactive SP (I-SP) in extracted plasma of the cat was 69.3 +/- 9.8 fmol/ml with values ranging from 2.5 to 165 fmol/ml. Evisceration of the cats caused a decrease of I-SP levels from 70.8 +/- 30.8 fmol/ml to 20.8 +/- 9.9 15 min and to 26.8 +/- 19.7 fmol/ml 60 min after the operation. 3. Ligation of intestinal blood vessels led to a fall in I-SP levels from 58.7 +/- 11.5 to 25.9 +/- 4.1 fmol/ml within 15 min. 4. No difference between I-SP values in portal (71.8 +/- 11.2 fmol/ml) and peripheral plasma (68.3 +/- 12.1 fmol/ml) was found under the conditions in which these experiments were performed. 5. It is concluded that a major part of circulating immunoreactive SP originates from the intestine.