We demonstrate the functional activity of single ribosomal complexes, opening the way for detailed studies of the trajectories of protein synthesis. Our approach employs a single-molecule detection system, capable of picoseconds to minutes resolution, to observe a growing peptide labeled at its N terminus with the fluorophore tetramethylrhodamine (TMR). Single complexes of mRNA-programmed ribosomes with TMR-Met-tRNAMetf or TMR-Met-Phe-tRNAPhe are immobilized on mica and observed by fluorescence. Immobilized ribosome.mRNA.TMR-Met-tRNAMetf complexes form peptide bonds with puromycin. Single-molecule detection reveals dynamics on the scale of seconds at the ribosomal peptidyl transferase center.