In humans there is a correlation between the ratio of arachidonic acid (20:4n-6) to cis 8,11,14 eicosatrienoic acid (20:3n-6) in skeletal muscle phospholipids and insulin sensitivity. This has been interpreted as indicating a link between the activity of the delta5 desaturase enzyme and muscle insulin sensitivity. The present study addressed the possibility that insulin regulates delta5 desaturase activity using L6 rat myoblasts and hepG2 human hepatoma cells. Both cell lines responded to insulin by increasing the amount of D-[U-14C] glucose incorporated into glycogen. In L6 cells, insulin stimulated cis 8,11,14 eicosatrienoic acid uptake and arachidonic acid production but had no effect on the percentage conversion of cis 8,11,14 eicosatrienoic acid to arachidonic acid. In hepG2 cells, insulin had no effect on cis 8,11,14 eicosatrienoic acid uptake or arachidonic acid production. These results suggest that insulin has no direct effect on delta5 desaturase activity in the liver but can alter arachidonic acid production in muscle by altering substrate availability.