In the transportation sector, users are taking a new look at natural gas (NG) as an affordable alternative, often priced at $1.50-2.00/gasoline gallon equivalent (GGE) less than gasoline or diesel, and offering potential societal benefits like reductions in petroleum use, air pollutant emissions, and greenhouse gas (GHG) emissions. Unlike petroleum, a significant portion of which is imported and vulnerable to external disruptions at key points in the supply chain, NG is almost completely domestic and supplies are diverse. NG from renewable sources like landfills and anaerobic digesters or from fossil sources like shale gas is found in nearly every state, with associated benefits for state economies. An extensive transmission and distribution infrastructure exists in much of the country. There are, however, significant barriers that limit the speed and breadth of NG uptake into the transportation sector. Many of the barriers to NG use in this sector are technical in nature and overcoming them will require a concerted R&D effort. This paper focuses on those barriers and associated R&D opportunities for using compressed natural gas (CNG), liquefied natural gas (LNG), and NG-derived liquid fuels in internal combustion engines (ICEs) for light duty vehicle (LDV) and medium- and heavy-duty truck (M/HDT) applications. This paper is organized into five key areas that highlight R&D needs: NG distribution, liquid fuel production from stranded gas, vehicle fuel use, NG storage, and impact analysis and assessment.