ABSTRACT To understand how the brain functions in health, and how sensory, motor, and cognitive functions are affected in disease, it is crucial to be able to record the activities of large numbers of individual neurons in real time. In the past two decades, calcium imaging in neuronal cell bodies has provided a conveniently qualitative view of neuronal activity, allowing action potential firing in specific neuron types or in various brain regions to be correlated with sensory input, decision-making, or internal representations of emotional or physiological parameters. However, somatic calcium is generally insensitive to subthreshold activity, i.e. to synaptic inputs that depolarize the membrane potential without eliciting action potentials, and lacks the temporal precision to determine the timing relationship between action potentials within a circuit. Our understanding of the brain would beneift greatly from understanding how neuronal circuits use transmembrane voltage to represent and process information. Outstanding questions include how neuronal types differ in their summatation of inputs to initiate an action potential output, how neuronal circuits extract salient features or makes a decision based on complex patterns of input activity, and how experience or neuromodulation or disease affects these processes. We propose to address this problem by creating a class of high-performance genetically encoded voltage indicators (GEVIs) to record both subthreshold and spiking activity in large numbers of neurons in living animals. In particular, we find that positively tuned GEVIs have the potential for detecting spikes with many times greater signal-to-noise ratio than GECIs while achieving useful discriminability of subthreshold potentials. We propose an intense effort to develop such positively tuned GEVIs toward ideal performance specifications identified by quantitative modeling. Aims include (1) comprehensive screening of residues in a prototype positively tuned GEVI to identify positions modulating voltage tuning and fluorescence responsiveness, followed by deep combinatorial mutagenesis of identified sites, (2) validation of indicators in vivo in 1-photon and 2-photon imaging in flies and mice, and (3) development of an ultra-high-throughput single-cell screening system to further accelerate GEVI improvement.