An optimized gene expression construct was designed in order to increase the accumulation of the HIV-1 subtype C p24 protein in Arabidopsis thaliana and carrot (Daucus carota) plants. An ER retention signal was introduced into the genetic construct generating a p24 protein containing a SEKDEL amino acid sequence at its C-terminus. Mature A. thaliana plants and carrot cells were transformed using Agrobacterium tumefaciens carrying the improved pGreen0229/p24_SEKDEL vector. Several transgenic plant lines were obtained from both plant species by growth on selective medium and confirmed by PCR. Transformed lines were analyzed for p24 protein content by western blotting using anti-p24-specific antibodies and by Southern blotting to establish the number of copies of the insert in the plant nuclear genome. To estimate the accumulation levels of p24 protein in the plants, ELISA was run using soluble plant extracts. By comparing these results with our previous findings, the ER retention signal increased the level of p24 protein fivefold in the A. thaliana plants. In carrot taproot, the content of p24_SEKDEL protein was approximately half of that in Arabidopsis on a fresh weight basis and was stable in planta for several months. However, on a total soluble protein basis, carrots produced considerable higher levels of the p24_SEKDEL protein than Arabidopsis.