The interaction of a series of chromophoric nucleotides derived from 6-mercapto-9-beta-ribofuranosylpurine (thioinosine, thiol) and 2-amino-6-mercapto-9-beta-ribofuranosyl-purine (thioguanosine, thioG) with myosin subfragment 1 isolated from rabbit skeletal muscle was investigated kinetically and spectroscopically. The Mg2+-dependent hydrolyses of thioITP and thioGTP are catalysed by subfragment 1 and probably proceed by a similar mechanism as for ATP hydrolysis, although with different rate constants. For example, the binary thioGDP-protein complex only comprises 8% of the steady-state intermediate of the thioGTPase at 5 degrees C and pH 6.5. Long-lived analogues of intermediates of the thioGTPase were generated by using thioGTP(gammaS) [thioguanosine 5'-(3-thio)-triphosphate], thioGMP-P(NH)P (5'-thioguanylylimidodiphosphate) and thioGDP. The near-u.v. spectra of the thioguanosine nucleotides bound to subfragment 1 were measured and showed that in all cases the purine ring is bound to the protein in a hydrophobic environment, although the pK of the purine thiol group only increases by 0.2-0.3. ThioGTP caused glycerinated rabbit psoas muscle to contract, but in contrast with thioITP was not able to relax muscle. The applications of these chromophoric nucleotides for investigating the mechanism of muscle contraction and other biological systems, particularly those involving guanosine nucleotide regulation, are discussed.