Understanding biology at the single-cell level requires simultaneous measurements of biochemical parameters and behavioral characteristics in individual cells. Here, the output of individual flagellar motors in Escherichia coli was measured as a function of the intracellular concentration of the chemotactic signaling protein. The concentration of this molecule, fused to green fluorescent protein, was monitored with fluorescence correlation spectroscopy. Motors from different bacteria exhibited an identical steep input-output relation, suggesting that they actively contribute to signal amplification in chemotaxis. This experimental approach can be extended to quantitative in vivo studies of other biochemical networks.