Photoreactive derivatives of imidacloprid and its nitromethylene analogue were synthesized as candidate photoaffinity probes for identifying the amino acid residues of nicotinic acetylcholine receptors (nAChRs) that interact with the neonicotinoid insecticides. When the candidate probes were injected into American cockroaches, the nerve cord neural activity initially increased, then ceased and death of the insect followed. Both the nerve cord and toxicity were enhanced by changing the photoreactive substituent from the para position to the meta position on the spacer benzyl moiety. When tested on a Drosophila SAD/chicken beta2 hybrid, recombinant nAChR expressed in Xenopus oocytes, the nitromethylene candidate probes showed agonist activity similar to that previously observed for imidacloprid.