Plants have long been used by humans as primary sources of food, medicine, fibre and energy. Over time these diverse applications have gradually expanded in scope, scale and sophistication, with the most recent advancement deriving from the development of biotechnology, resulting in previously unimagined enhancements in plant productivity and quality. Recently, the increasing concern about declining global supplies of fossil oil from which we derive transportation fuels and a wide range of chemical feedstocks has generated new and urgent interest in expanding the applications of plants so as to produce alternatives to those materials. It is also argued that supplementing or replacing petroleum-based fuels and petrochemicals with agriculturally produced, renewable equivalents is appealing from an environmental standpoint. While the development of plant-based solutions to this challenge includes traditional plant selection and breeding, along with applications research, we also anticipate a significant ‘investment’ by molecular plant biotechnology. Indeed, plant biotechnology may prove to be an essential tool in developing plants with new properties for use in the most efficient production of transportation biofuels and phytochemicals. The considerable chemical energy contained in harvested plant biomass of various kinds (wood, seed oils, waste stems and foliage etc.) has long been used as a source of useful thermal, mechanical and electrical energy via conversion which relies on the simple process of ‘external combustion’. Thus there is little doubt that, theoretically, plants could prove to be productive sources of energy for today’s transportation needs. However, the challenge presented in supplementing petroleum for its major uses in modern transportation is a considerable one, as these applications are heavily reliant on conversion of chemical to mechanical energy in ‘internal combustion’ processes which are only compatible with liquid or gaseous fuels (spark-ignition, compression-ignition and turbine engines). The deployment of plant biomass as transportation biofuels must therefore involve not only cost-effective agricultural production on substantial land acreages with minimal inputs, but also efficient chemical conversion into liquid- or gaseous-phase compounds which retain adequate calorific value and other engine-compatible properties, and which are themselves readily transported (piped or hauled in tanks). These specifications will only be achieved through the development and optimization of a series of plant characteristics that range all the way from overall ‘agronomic’ traits (growth habit, compatibility with production and harvesting methods, stress tolerance, input efficiencies, yield etc.), to molecular-level modifications in constituents such as lignocellulose that will ensure optimal performance in the postharvest conversion process, and perhaps to the presence or absence of certain secondary metabolites that affect performance of the end-product. A particularly attractive aim is the development of plants that can be grown for these purposes on land that is not currently used for food, thus avoiding competition between food and energy production. Given the diversity and urgency of these essential modifications, plant biotechnology surely has a major role to play in the development of successful ‘biofuel’ crops, hence our choice of this topic for the second Special Issue of Plant Biotechnology Journal. The first especially dedicated issue of Plant Biotechnology Journal was published in May 2009. Its contributors reviewed advances in single nucleotide polymorphism (SNP) analysis (Henry and Edwards, 2009), a technology applicable to the development of plants for all applications. This second Special Issue features applications of plant biotechnology in the development of plants that will generate biofuels. Issues discussed include manipulation of the biosynthesis of carbohydrate and lignin components of plant biomass and technologies available for conversion of plant biomass to fuel including ‘in planta’ production of enzymes to aid this conversion. Progress in developing plants for use in production of high-value products will be the subject of a future Special Issue.