Twenty-nine hammerhead-like RNA sequences have been synthesized and analyzed for cleavage activity. Each of the 29 sequences contains a portion of the hammerhead ribozyme RNA sequence that includes the sequence-recognizing helices stem I and stem III and most of the conserved catalytic core sequence, but loop II and stem II have been replaced fully or in part by non-nucleoside linker molecules. In some complexes, the nonconserved core nucleoside U7 has also been replaced by appropriate linker molecules. The non-nucleoside linkers include hexaethylene glycol, triethylene glycol, 1,3-propanediol, and an abasic site analogue based upon tetrahydrofuran, All complexes prepared were analyzed under single turnover conditions, and first-order rate constants were obtained to characterize the cleavage reactions. Complexes exhibiting native-like cleavage efficiency were further analyzed under steady-state conditions and were characterized by Michaelis-Menten parameters (K-m and k(cat)). The results indicate that the four base pairs that constitute loop II can be removed without any significant change-in cleavage activity. Loop II and two base pairs from stem II can also be removed, but the activity of these stem-shortened ribozymes is highly dependent upon the sequence of the remaining two base pairs. A two-base pair stem containing the bases G(10.1)-C-11.1, C-10.2-G(11.2) exhibited cleavage activity that was very similar to that of the native complex, while the other three possible stem sequences containing G-C base pairs were all reduced in activity by at least 2 orders of magnitude. Replacement of the nonconserved core nucleoside, U7, with linker molecules did not result in RNA catalysts with remarkable cleavage activity.