Objective: This study sought to identify and prioritise research issues as perceived by Australian general practice nurses. In this context, a research priority refers to the most pressing research problems that necessitate exploration to improve clinical practice. Design: This paper reports the findings of a two-round Delphi study. Initially, focus groups identified research issues. Subsequently, an online survey facilitated ranking of these issues on a 5-point Likert scale. Setting: Australian general practices. Subjects: Twenty-five practice nurses participated in the focus groups and 145 practice nurses responded to an online survey. Main outcome measure(s): The main outcome measure was the prioritisation of research issues by practice nurses. Results: The focus groups identified 53 research issues. These could be broadly classified as issues related to: (1) the practice nurse role and professional development; (2) general practice service delivery; and (3) disease prevention and health promotion. Survey data identified 34 priority areas from the 53 issues, that were considered important as indicated by a mean score greater than 4.0. These included 17 items related to disease prevention and health promotion, 12 items related to the practice nurse role and professional development and five items related to general practice service delivery. Conclusions: Issues identified in this study reflect current patterns of practice nurse workload. Emphasis needs to be placed on the translation and use of evidence by practice nurses, as well as the ways in which evidence can support and promote development of their role. The study findings highlight the need for research that is responsive to clinical demands.