Phosphatidylinositol 3,4,5-trisphosphate (PIP3) plays an important role in the regulation of diverse physiological functions. Recent evidence indicates that PIP3 is cell permeant, and can be added exogenously to modulate cellular responses. However, like many other phospholipids, PIP3 binds serum proteins with high affinity, resulting in rapid deactivation of this lipid second messenger. Our study indicates that bovine serum albumin (BSA) at concentrations as low as 10 μg/mL abrogated the biological activity of dipalmitoyl-PIP3. This nonspecific interaction with serum proteins hampers the use of PIP3 in biological studies where serum is needed. We report here an ether-linked PIP3 analogue, 1-O-(1-O-hexadecyl-2-O-methyl-sn-glycero-3-phosphoryl)-myoinositol 3,4,5-trisphosphate (C16Me-PIP3), which displays low serum protein-binding affinity while retaining the biological function of PIP3. The affinity of C16Me-PIP3 with BSA was two orders of magnitude lower than that of its dipalmitoyl-counterpart. Biochemical data indicate that C16Me-PIP3 was able to stimulate Ca2+ influx in T cells in the presence of moderate levels (up to 1 mg/mL) of BSA. Thus, C16Me-PIP3 may provide a useful tool to study the physiological function of phosphoinositide (PI) 3-kinase in vivo.