BACKGROUND: Small interfering RNAs (siRNAs) can induce specific gene silencing through cytoplasmic mRNA cleavage and nuclear transcriptional silencing, necessitating delivery to different cellular compartments. This study presents a reducible copolypeptide (rCPP) carrier containing different molar ratios of a histidine-rich peptide (HRP) and nuclear localization sequence (NLS) peptide to modulate intracellular trafficking of transfected siRNA and primary RNA transcripts (pri-miRNA). METHODS: Polyplex formation using siRNA and rCPP was demonstrated using photon correlation spectroscopy and atomic force microscopy. Confocal and fluorescence microscopy were used to investigate cellular uptake and nuclear trafficking whilst endogenous enhanced green fluorescent protein (EGFP) knockdown in H1299 cells was evaluated using flow cytometry. Transcriptional gene silencing of endogenous EF1A was verified using real-time reverse-transcription polymerase chain reaction (RT-PCR) and pri-miRNA nuclear processing was demonstrated using Northern analysis. RESULTS: rCPP-based polyplexes showed rapid cellular uptake and low cytotoxicity. Labelled components revealed intact polyplexes after 2 h that exhibited directed movements consistent with endosomal trafficking. Polyplex-mediated knockdown of EGFP increased with greater HRP content. The inclusion of NLS promoted nuclear localization of transfected siRNAs and pri-miRNAs to the nuclear compartment allowing for transcriptional silencing of EF1A and Drosha and Dicer dependent expression of mature miRNA, respectively. CONCLUSION: Our results demonstrate that reducible copolypeptides can be used as carriers for the non-toxic cellular delivery of siRNA and pri-miRNA. The nuclear targeting of rCPPs can be utilized for delivery of siRNAs and pri-miRNAs to the nuclear compartment for transcriptional gene silencing or endogenous processing into mature miRNA, respectively, which could potentially lead to improved therapeutic approaches.