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Dual-color photometry revealed that dopamine release in the nucleus accumbens evoked by reward-predictive cues is accompanied by a rapid suppression of glutamate release. We demonstrate the utility of RdLight1 for receptor-specific pharmacological analysis in cell culture, simultaneous assessment of dopamine release and cell-type-specific neuronal activity and simultaneous subsecond monitoring of multiple neurotransmitters in freely behaving rats. RdLight1 can be combined with GFP-based sensors with minimal interference and shows high photostability, permitting prolonged continuous imaging. We therefore engineered red-shifted variants of dopamine sensors called RdLight1, based on mApple. However, these GFP-based variants cannot be readily combined with commonly used optical sensors and actuators, due to spectral overlap. encoded dopamine sensors based on green fluorescent protein (GFP) enable high-resolution imaging of dopamine dynamics in behaving animals. 12 Department of Biochemistry and Molecular Medicine, University of California, Davis, Sacramento, CA, USA. 11 Weill Institute for Neurosciences and Kavli Center for Fundamental Neuroscience, University of California, San Francisco, San Francisco, CA, USA. 10 Department of Neurology, University of California, San Francisco, San Francisco, CA, USA. 9 Weill Institute for Neurosciences and Kavli Center for Fundamental Neuroscience, University of California, San Francisco, San Francisco, CA, USA. 8 Department of Psychiatry, University of California, San Francisco, San Francisco, CA, USA.1 Department of Biochemistry and Molecular Medicine, University of California, Davis, Sacramento, CA, USA. 7 Department of Psychiatry, University of California, San Francisco, San Francisco, CA, USA.6 Key Laboratory of Medical Electrophysiology, Institute of Cardiovascular Research, Southwest Medical University, Luzhou, China.5 Center for Neuroscience, University of California, Davis, Davis, CA, USA.4 Department of Neurology, University of California, San Francisco, San Francisco, CA, USA.3 Neuroscience Center Zurich, University and ETH Zurich, Zurich, Switzerland.2 Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland.1 Department of Biochemistry and Molecular Medicine, University of California, Davis, Sacramento, CA, USA.