Several recent studies have used antisense oligonucleotides in the nervous system to probe the functional role of particular gene products. Since antisense oligonucleotide‐mediated block of gene expression typically involves uptake of the oligonucleotides, we have characterized the mechanism of this uptake into developing neurons from embryonic chickens. Antisense oligonucleotides (15 mers) added to the bathing media are taken up into the embryonic chicken sympathetic neurons maintained in vitro. A portion of the oligonucleotide uptake is temperature dependent and saturates at extracellular oligonucleotide concentrations ≥ 20 μM. This temperature sensitive, saturable component is effectively competed by single nucleotides of ATP and AMP and is reminiscent of receptor‐mediated endocytosis of oligonucleotides described in non‐neuronal cells. The efficiency of the oligonucleotide uptake system is dependent on the developmental stage of the animal but independent of the number of days that the neurons are maintained in vitro. Following the uptake of antisense oligonucleotides directed against ion channel subunit genes expressed by these neurons (nicotinic acetylcholine receptor subunit α3; nAChR α3), biophysical assays reveal that the functional expression of the target gene is largely blocked. Thus the number of wild type nAChR channels expressed is decreased by =80%–90%. Furthermore, following antisense deletion of α3, “mutant” nAChRs with distinct functional characteristics are expressed. In sum, these studies characterize the uptake of antisense oligonucleotide and demonstrate the functional block of specific gene expression in primary developing neurons. In addition, the functional studies emphasize the need for sensitive and specific assay following antisense deletion, since other homologous gene products may substitute for the targeted gene resulting in new phenotypes that are subtly different from wild type. © 1993Wiley‐Liss, Inc. Copyright © 1993 Wiley‐Liss, Inc.