The involvement of ecto-phosphoryl transfer transduction in the ATP induced contraction of the vas deferens was examined. Vas deferens obtained from English-short-hair-guinea-pigs were treated with 10(- 2) molar (M) periodate-oxidized-ATP (P-ATP), an inhibitor of the second phase of the vas deferens contraction to ATP, or 10(-4)M arylazido-aminopropionyl-ATP (ANAPP3), an inhibitor of the first phase of the contraction. Control preparations were treated with saline. Intact and homogenized vas deferens were then incubated with 3x10(-3)M phosphorus (32P) radiolabeled ATP or sulfur (35S) radiolabeled adenosine-5'-O-(3-thiotriphosphate) (ATP-gamma-S) for 5 to 60 seconds. The tissues were analyzed using sodium-dodecyl- sulfate polyacrylamide-gel electrophoresis (SDS/PAGE). Controls were incubated with the radiolabel only. Vas deferens incubated with 10(-2)M radiolabeled P-ATP for 5 minutes were also subjected to SDS/PAGE. Intact vas deferens incubated with radiolabeled ATP-gamma- S or radiolabeled ATP incorporated 35S or 32P, respectively, into a 19 to 21 kilodalton (kD) protein, at an intensity which corresponded to the time course of the contraction. Thus, 35S labeling occurred within 5 seconds, peaked within 10 seconds, and decreased significantly thereafter. The incorporation of 32P peaked within 5 seconds and decreased significantly thereafter. The incorporation of 35S was not observed in homogenized tissue. Treatment with ANAPP3 caused a significant decrease in 35S incorporation at 60 seconds. Treatment with P-ATP induced significant decreases in 35S incorporation at 60 seconds in intact tissue and 5 seconds in homogenized tissue. Treatment with P-ATP had no effect on 32P incorporation. In intact vas deferens, radiolabeled P-ATP was incorporated into several proteins, not including the 19 to 21kD protein. The presence of excess ATP enhanced 35S incorporation and inhibited P-ATP labeling. Neither 35S nor P-ATP labeling were affected by either norepinephrine or histamine. The authors conclude that an ecto-phosphoryl transfer transduction is involved in the second phase of contraction to ATP.