The interaction of aspartate transcarbamylase from Escherichia coli with the activator 6-mercapto-9-β-D-ribo-furanosylpurine 5′-triphosphate (sRTP) has been investigated at pH 7.0, 25°, in 0.15 M potassium acetate-0.04 M imidazole acetate using difference spectroscopy and the temperature-jump method. The sRTP does not serve as an affinity label for the regulatory or catalytic sites, but a difference spectrum is observed when sRTP binds to the catalytic subunit, the regulatory subunit and the native enzyme. A spectral titration of the catalytic subunit indicates three binding sites are present per catalytic subunit molecule with a dissociation constant of 2.5 × 10-4 M. Although a difference spectrum also accompanies binding to the regulatory subunit, the binding is too weak for quantitative characterization. In the absence of substrates, the difference spectrum of the sRTP-native enzyme interaction is very similar to that found for the catalytic sub-unit, but the addition of 2 mM carbamyl phosphate shifts the maximum in the difference spectrum from 332 to 325 nm. Further addition of the aspartate analog, succinate, decreases the absolute magnitude of the difference spectrum, but does not shift the wavelength maximum. The binding of sRTP to the isolated catalytic subunit gives rise to a temperature-jump relaxation process which is too fast and of too low an amplitude for detailed study. With native enzyme, two relaxation processes are seen. The faster one has a time constant similar to that found with the isolated catalytic subunit and disappears in the presence of 2 mM carbamyl phosphate so that it probably reflects the interaction of sRTP with the catalytic site. The reciprocal relaxation time for the slower process increases and approaches a constant value as the sRTP concentration is raised. This behavior is observed in the presence or absence of 2 mM carbamyl phosphate and 10 mM succinate, although the limiting value reached varies. The simplest mechanism consistent with the data is a rapid combination of sRTP and enzyme followed by a rate-limiting conformational change, a mechanism similar to that proposed for the interaction of cytidine 5′-triphosphate with the native enzyme. When both sRTP and 5-bromocytidine 5′-triphosphate are added to the enzyme, only a single relaxation process is observed suggesting that the same two conformational states occur with both activator and inhibitor complexes. Carbamyl phosphate and succinate and sRTP tend to stabilize one conformational state, while cytidine 5′-triphosphate stabilizes the other. A different conformational transition accompanies the binding of succinate to the native enzyme; sRTP decreases the reciprocal relaxation time of this process as its concentration is raised, while 5-bromocytidine 5′-triphosphate has the opposite effect. A multiconformational model involving both concerted and sequential conformational transitions is proposed for the overall regulatory mechanism.