Olomoucine is known as a cyclin-dependent kinase inhibitor. We found that olomoucine blocked insulin's ability to stimulate glucose transport. It did so without affecting the activity of known insulin signaling proteins. To identify the olomoucine-sensitive kinase(s), we prepared analogs that could be immobilized to an affinity resin to isolate binding proteins. One of the generated analogs inhibited insulin-stimulated glucose uptake with increased sensitivity compared with olomoucine. The IC₅₀ for inhibition of insulin-stimulated glucose uptake occurred at analog concentrations as low as 0.1 µM. To identify proteins binding to the analog, [³⁵S]-labeled cell lysates prepared from 3T3-L1 adipocytes were incubated with analog chemically cross-linked to a resin support, and binding proteins analyzed by SDS-PAGE. The major binding species was a doublet at 50-60 kDa, which was identified as CaMKII by N terminal peptide analysis, and confirmed by MALDI-MS as the and -like isoforms. To investigate CaMKII involvement in insulin-stimulated glucose uptake, 3T3-L1 adipocytes were infected with retrovirus encoding GFP-HA-tagged CaMKII wild-type or the ATP binding mutant, K42M. GFP-HA-CaMKII K42M cells had less kinase activity than cells expressing wild-type GFP-HA-CaMKII. Insulin-stimulated glucose transport was significantly decreased (80%) in GFP-HA-CaMKII K42M cells compared with non-transfected cells, and cells expressing either GFP-HA-CaMKII or GFP-HA. There was not a concomitant decrease in insulin-stimulated GLUT4 translocation in GFP-HA-CaMKII K42M cells when compared with GFP-HA alone. However, insulin-stimulated GLUT4 translocation in GFP-HA-CaMKII cells was significantly higher compared with either GFP-HA or GFP-HA-CaMKII K42M cells. Our results implicate the involvement of CaMKII in glucose transport in a permissive role.