Abstract Purpose. The purpose was to study the reduction of intra-canal microflora in premolars with apical periodontitis instrumented with either apical box or apical cone preparation and to provide measurements of intervention effects to allow proper power calculation in future clinical trials. Methods. Twenty-four patients were centrally randomized to apical box preparation (size #60) or cone preparation (apical size #25). The groups were comparable regarding the presence of primary caries and type of coronal restoration. In the course of canal preparation each tooth was irrigated with 2.5% NaOCl (12 ml). Lastly, the canals were filled with 17% EDTA (2 × 30 s) and 5% iodine potassium iodide (IKI) for 10 min. The canals were sampled for micro-organisms on four occasions: before instrumentation, after instrumentation, after application of IKI dressing and at the beginning of the second appointment 1 week later. Between the treatment sessions, the root canals were sealed with IRM cement. In the laboratory, culture techniques were used to measure microbial growth, which was classified as: none, very sparse, sparse, moderate, heavy or very heavy. Results. Initially, microbes were recovered in 88% of the teeth. Growth was classified as none in 35% of the teeth after instrumentation and in 50% after the application of IKI. Irrespective of the time of sampling, no significant difference in microbial growth reduction was observed between the two types of apical preparation. Based on the 1-week post-sampling, a power calculation revealed that over 900 patients are needed to show a difference of 9% between the two protocols tested. Conclusions. Future trials should be conducted using stringent protocols and as multi-centre trials for reaching the required information size.