The antibody 38C2 efficiently catalyzed a retro-Michael reaction to convert a novel, cell-permeable fluorogenic substrate into fluorescein within living cells. In vitro, the antibody converted the substrate to fluorescein with a k(cat) of 1.7 x 10(-5) s(-1) and a catalytic proficiency (k(cat)/k(uncat)K(m)) of 1.4 x 10(10) m(-1) (K(m) = 7 microm). For hybridoma cells expressing antibody or Chinese Hamster Ovarian (CHO) cells injected with antibody, incubation of the substrate in the extracellular medium resulted in bright intracellular fluorescence distinguishable from autofluorescence or noncatalyzed conversion of substrate. CHO cells loaded with antibody were 12 times brighter than control cells, and more than 85% of injected cells became fluorescent. The fluorescein produced by the antibody traveled into neighboring cells through gap junctions, as demonstrated by blocking dye transfer using the gap junction inhibitor oleamide. The presence of functional gap junctions in CHO cells was confirmed through oleamide inhibition of lucifer yellow transfer. These studies demonstrate the utility of the intracellular antibody reaction, which could generate tracer dyes in specific cells within complex multicellular environments simply by bathing the system in substrate.