The K(v)2.1 potassium channel plays an important role in regulating membrane excitability and is highly phosphorylated in mammalian neurons. Our previous results showed that variable phosphorylation of K(v)2.1 at multiple sites allows graded activity-dependent regulation of channel gating. Our previous studies also found functional differences between recombinant K(v)2.1 channels expressed in HEK293 cells and COS-1 cells that were eliminated upon complete dephosphorylation of K(v)2.1. To better understand how phosphorylation affects K(v)2.1 gating in HEK293 and COS-1 cells we used stable isotope labeling by amino acids in cell culture (SILAC) and mass spectrometry to determine the level of phosphorylation at one newly and thirteen previously identified sites on K(v)2.1 purified from HEK293 and COS-1 cells. We identified seven phosphorylation sites on the K(v)2.1 C-terminus that exhibit different levels of phosphorylation in HEK293 and COS-1 cells. Six sites have enhanced phosphorylation in HEK293 compared to COS-1, while one site exhibits enhanced phosphorylation in COS-1 cells. No sites were found phosphorylated in one cell type and not the other. Interestingly, the sites exhibiting differential phosphorylation in HEK293 and COS-1 cells under basal conditions are similar to the subset targeted by calcineurin-mediated signaling pathways. The data presented here suggests that differential phosphorylation at a specific subset of sites, as opposed to utilization of novel cell-specific phosphorylation sites, can explain differences in the gating properties of K(v)2.1 in different cell types under basal conditions, and in the same cell type under basal versus stimulated conditions.