An important challenge in antisense technology remains the adequate delivery of the oligonucleotides (ON) to individual cells. Understanding the subcellular distribution of ONs and their carrier is essential to explain the (lack of) biological activity. The ability of several cationic carriers to efficiently deliver anti-ICAM-1 oligonucleotides to their site of action was studied using a cell-based assay. In this assay we evaluated the ability of the ONs to downregulate the expression of the ICAM-1-protein in A549 cells. To understand why some carrier/ONs combinations showed biological activity while others failed, flow cytometry and confocal laser scanning microscopy (CLSM) measurements were used to study cellular uptake and intracellular distribution of the (fluorescently labeled) ONs. We showed that free ONs (both PS-ONs and PO-ONs) and ONs complexed to pEGpEI failed to decrease the ICAM-1 protein level. This was due to the inability of the (free or complexed) ONs to enter the cell, as shown by flow cytometry and CLSM. Flow cytometry and CLSM showed cellular uptake when PO-ONs and PS-ONs were complexed to graft-pDMAEMA and Lipofectin. However, while the uptake and intracellular localization seemed similar for ONs complexed to, respectively, graft-pDMAEMA and Lipofectin, the biological activity of the ONs was clearly dependent on their carrier: both PO-ONs and PS-ONs complexed to graft-pDMAEMA reduced the ICAM-1 expression; however, when complexed to Lipofectin only PS-ONs showed biological activity. Also, PS-ONs complexed to graft-pDMAEMA were more active than PO-ONs complexed to graft-pDMAEMA which could not be explained by the results from CLSM and flow cytometry. While the ICAM-1 assay proves whether a certain pharmaceutical carrier successfully delivers ONs or not, it does not answer the important question why one carrier is successful while another one fails. Also, our study shows that flow cytometry and CLSM, although useful techniques, failed to clearly explain the difference in transfection behavior between graft-pDMAEMA and Lipofectin. As ONs become susceptible to degradation by cytosolic DNase as soon as they are released from their carrier, one could argue that a better understanding of the time and (intracellular) place at which the dissociation of the complexes occurs could be crucial to fully explain our observations.