Uptake of Na275SeO3 by mitochondria of the larvae of the insect C. cephalonica reared at different dietary selenium (Se) levels revealed: 1. A proportional increase in the uptake with externally added Na275SeO3 in concentrations upto 25.32 μM; and 2. At each added selenite concentration, an increase up to 60 min, with linearity up to 15-30 min. A differential affinity for Na275SeO3 was elicited in the mitochondrial protein fractions of different dietary Se groups and correlated well with the pattern and the ratio of distribution of incorporated75Se in protein to nonprotein fractions. Kinetic studies on75Se uptake by whole mitochondria negated passive diffusion of selenite and revealed a trend of negative cooperativity confirmed by Hill and Scatchard plots. Half saturation value was estimated to be approx 13 nmole Se/mg mitochondrial protein. Scatchard plot for75Se uptake was biphasic and the high affinity binding sites were estimated to be around 5 nmole/mg mitochondrial protein. Calculated dissociation constants revealed maximal affinity for75Se in the 1.5 ppm group (KSe 0.0034 n M) and minimal in the basal group (KSe 0.007 n M). In the mitochondria of all the three dietary Se groups, the estimated low affinity sites amounted to be 15-19 nmole/mg mitochondrial protein. Inherent Se in the mitochondria of the high Se group positively enhanced the incorporation of75Se in the mitochondrial protein fraction. About 20-30% of the total uptake was indicated to be energy linked as revealed by studies with respiratory inhibitors. Addition of sulfite and sulfate (5-25 μM) in the medium, inhibited75Se uptake by 35-55%, suggestive of the involvement of the dicarboxylate port. Thiol interactive75Se uptake was confirmed by the inhibition mediated by mersalyl and NEM up to 50-70%. The study revealed thiol-selenite interactions of metabolic significance during selenite uptake. © 1995 Humana Press Inc.