Recent global flu pandemic highlights the obvious need for a vaccine against the influenza virus that is both inexpensive to produce and can be produced fast enough for development on a large scale. The influenza virus coat protein hemagglutinin (HA) is a major surface antigen and has emerged as a good candidate antigen for subunit vaccine development. Previous attempts to express HA from the A/Sichuan/2/87 H3N2 influenza subtype in plastids achieved HA gene transcription but no protein accumulation, suggesting that the HA protein may be unstable in the plastid. The aim of this work is to investigate alternative strategies that might achieve HA antigen accumulation in plastids. Two series of gene constructs were made. The first series of constructs coded for the first 14 amino acids of GFP and RBCL N-terminally fused to the full length HA, a full length GFP N-terminally fused to HA and a full length GFP N-terminally fused to HA1. The second series of gene constructs coded for two candidate influenza virus hemagglutinin epitopes, HA91-108 and HA 307-319 fused singly as N and C terminal fusions and as double terminal fusions to full length GFP. A total of ten gene constructs were introduced into the chloroplast genome by biolistic bombardment. Transformants were generated and confirmed through molecular analyses. Transplastomic lines generated with the 14aaRBCL/HA and 14aaGFP/HA constructs accumulated a recombinant protein that was smaller than expected. Further analysis is required to confirm that these proteins are indeed truncated HA fusion proteins. No transformed plant lines were generated with the full length GFP/HA and GFP/HA1 constructs suggesting that the tobacco plastid is not a useful system for producing full-length flu virus HA and HA1 proteins suitable for vaccine development. Expression and stability of recombinant HA91-108/GFP and GFP/HA307-319 fusion proteins was clearly demonstrated showing that conserved influenza virus HA epitopes can be expressed in plant chloroplasts. No transgene transcripts could be detected in transplastomic lines generated with the GFP/HA91-108, HA307-319/GFP or HA307-319/GFP/HA91-108 gene constructs. No transformed lines were generated with the HA91-108/GFP/HA307-319 construct. Conditions for shoot regeneration and the parameters required for biolistic-mediated chloroplast transformation of lettuce were also assessed. However, despite numerous attempts, no plastid transformed lettuce shoots were obtained.