Broadly applicable strategies facilitating direct and selective modulation of the intracellular levels of physiologically important small molecules are essential for dissecting their integral and multiple roles in cellular processes. Therefore, we have been exploring the suitability of RNA aptamers for this purpose. Using the Escherichia coli heme biosynthetic pathway as a simple model of a negative feedback regulated process, we show that heme-binding RNA aptamers, developed in vitro and expressed intracellularly, induce a heme-dependent growth defect in an E. coli heme auxotroph defective in converting delta-aminolevulinic (delta-ALA) acid into downstream products. Relative to a control oligonucleotide, the aptamers also induce delta-ALA accumulation in cells grown under heme-limiting conditions. Increasing the concentration of heme in the media completely reverses both the growth defect and delta-ALA accumulation, except for two aptamers for which reversal is partial. Thus, these aptamers specifically target their cognate ligand in vivo and functionally modulate its intracellular concentration, demonstrating that RNA aptamers are useful tools for elucidating the role of heme and possibly other small molecules in regulating cellular networks.