The GIR1 branching ribozyme constitutes a separate class of naturally occurring ribozymes. Most studies have been performed with the single GIR1 known from the myxomycete Didymium iridis whereas the large number of GIR1s found in the amoeboflagellate Naegleria has remained largely uncharacterized. Here, we investigate ribozyme cleavage properties of a collection of Naegleria GIR1 ribozymes and define the variant from N. pringsheimi as a suitable model due to its superior activity in vitro. We identify the minimal ribozyme by deletion analysis applying a new RNase R based assay for the branching reaction, and by mutational analysis we demonstrate a surprising effect on the activity of structural elements J2/10 and L9 located outside the core of the ribozyme. These elements are located in regions that differ mostly from the Didymium ribozyme and illustrate the usefulness of comparative ribozyme studies.