A B STR ACT The effects of laser-flash photolytic release of ATP from caged ATP [P3-1(2-nitrophenyl)ethyladenosine-5'-triphosphate] on stiffness and tension transients were studied in permeabilized guinea pig portal vein smooth muscle. During rigor, induced by removing ATP from the relaxed or contracting muscles, stiffness was greater than in relaxed muscle, and electron microscopy showed cross-bridges attached to actin filaments at an -45 ~ angle. In the absence of Ca 2+, liberation of ATP (0.1-1 mM) into muscles in rigor caused relaxation, with kinetics indicating cooperative reattachment of some crossbridges. Inorganic phosphate (Pi; 20 mM) accelerated relaxation. A rapid phase of force development, accompanied by a decline in stiffness and unaffected by 20 mM P~, was observed upon liberation of ATP in muscles that were released by 0.5-1.0% just before the laser pulse. This force increment observed upon detachment suggests that the cross-bridges can bear a negative tension. The second-order rate constant for detachment of rigor cross-bridges by ATP, in the absence of Ca 2+, was estimated to be 0.1-2.5 x 105 M-~s -~, which indicates that this reaction is too fast to limit the rate of ATP hydrolysis during physiological contractions. In the presence of Ca "+, force development occurred at a rate (0.4 s -~) similar to that of intact, electrically stimulated tissue. The rate of force development was an order of magnitude faster in muscles that had been thiophosphorylated with ATP~,S before the photochemical liberation of ATP, which indicates that under physiological conditions, in non-thiophosphorylated muscles, light-chain phosphorylation, rather than intrinsic properties of the actomyosin cross-bridges, limits the rate of force development. The release of micromolar ATP or CTP from caged ATP or caged CTP caused force development of up to 40% of maximal active tension in the absence of Ca 2+, consistent Address reprint requests to Dr. Avril V. Somlyo,