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Background Rubisco (Ribulose-1,5-bisphosphate carboxylase/oxygenase) is a Calvin Cycle enzyme involved in CO2 assimilation. It is thought to be a major cause of photosynthetic inefficiency, suffering from both a slow catalytic rate and lack of specificity due to a competing reaction with oxygen. Revealing and understanding the engineering rules that dictate Rubisco's activity could have a significant impact on photosynthetic efficiency and crop yield.Results This paper describes the purification and characterisation of a number of hydrophobically distinct populations of Rubisco from both Spinacia oleracea and Brassica oleracea extracts. The populations were obtained using a novel and rapid purification protocol that employs hydrophobic interaction chromatography (HIC) as a form I Rubisco enrichment procedure, resulting in distinct Rubisco populations of expected enzymatic activities, high purities and integrity. Conclusions We demonstrate here that HIC can be employed to isolate form I Rubisco with purities and activities comparable to those obtained via ion exchange chromatography (IEC). Interestingly, and in contrast to other published purification methods, HIC resulted in the isolation of a number of hydrophobically distinct Rubisco populations. Our findings reveal a so far unaccounted diversity in the hydrophobic properties within form 1 Rubisco. By employing HIC to isolate and characterise Spinacia oleracea and Brassica oleracea, we show that the presence of these distinct Rubisco populations is not species specific, and we report for the first time the kinetic properties of Rubisco from Brassica oleracea extracts. These observations may aid future studies concerning Rubisco's structural and functional properties. [ABSTRACT FROM AUTHOR]