Colicins E1, E2, and E3 were covalently attached to Sephadex G-25 beads by cyanogen bromide activation. These immobilized colicins were still active in binding to specific receptors on sensitive and tolerant cells but not to resistant cells which lack such receptors. Bound colicin E3 also retained its ability to inhibit protein synthesis in vitro. Leakage of free colicin from these coated beads was negligible. Assays sensitive to free colicin activity of 1 part in 10(7) of the bound toxin failed to detect any soluble activity. The viability of different cell types bound specifically onto these colicin-Sephadex beads was assayed by using autoradiography based on labeled amino acid uptake. Immobilized E1 killed 90% of bound sensitive cells while less than 10% of sensitive cells bound to E2 and E3 were killed in this assay. These observations agree very well with previously suggested mechanisms which propose that E1, whose target site appears to be at the membrane level, can kill sensitive cells by binding to the cell surface, but that for E2 and E3 penetration of part or all of the molecule is necessary for killing action to be observed.