Apoptosis of pericytes (PCs) is an early event in diabetic retinopathy. It is generally thought to be a consequence of sustained hyperglycemia. In keeping with this, long-term (>7 days) incubation of cultured PCs in a high-glucose media has been shown to increase apoptosis. We examine here whether the saturated free fatty acid palmitate, the concentration of which is often elevated in diabetes, has similar effects on cultured PCs. Incubation with 0.4 mmol/l palmitate for 24 h induced both oxidant stress and apoptosis, as evidenced by a sixfold increase in DCF fluorescence and a twofold increase in caspase-3 activation, respectively. NAD(P)H oxidase appeared to be involved in these responses, since overexpression of dominant-negative subunits of NAD(P)H oxidase, such as phox47(DN), diminished oxidant stress, and phox67(DN) and N-17 RAC1(DN) prevented the increase in caspase-3 activity. Likewise, overexpression of vRAC, a constitutively active RAC1, increased caspase-3 activity to the same extent as palmitate alone. The effects of vRAC and palmitate were not additive. In parallel with the increases in oxidative stress, the redox-sensitive transcription factor nuclear factor-kappaB (NF-kappaB) was activated in cells incubated with 0.4 mmol/l palmitate. Furthermore, inhibition of NF-kappaB activation by various means inhibited caspase-3 activation. Finally, incubation with palmitate increased the cellular content of ceramide, a molecule linked to apoptosis and increases in oxidative stress and NF-kappaB activation in other cells. In keeping with such a role, in PCs both coincubation with fumonisin B1 (a ceramide synthase inhibitor) and overexpression of ceramidase I reversed the proapoptotic effect of palmitate. On the other hand, they increased rather than decreased DCF fluorescence. In conclusion, the results suggest that palmitate-induced apoptosis in PCs is associated with activation of NAD(P)H oxidase and NF-kappaB and an increase in ceramide. The precise interactions between these molecules in causing apoptosis and the importance of oxidant stress as a contributory factor remain to be determined.