Background: The class Mollicutes are minimal genomic prokaryotes with 23-28% GC content and a limited number of genomic tRNA species to service the production of proteins. As a result, the usage of GC-rich codons are much lower than GC-poor codons, and unique to Mollicutes is the use of the UGA codon (a stop codon in other species) as the preferential tryptophan codon compared to the single UGG codon available to other prokaryotes. Mollicutes are also notoriously small and very difficult to visualise in vitro or for in vivopathogenicity studies. Previously we have generated a successful site-directed mutagenesis method to deliver a gentamycin selection gene randomly into the Ureaplasma parvum genome. Here we extend those studies to deliver an optimised exogenous gene that enables visualisation of living Ureaplasma and other Mycoplasmas under the microscope and using a charged-coupled device (CCD) camera to view in vivo chemiluminescence. Method:The pMT85-based mini-transposase was utilised to deliver synthetic red fluorescent protein (RFP) or luciferase (luc) genes that were designed in silico for gene synthesis by Genscript ltd. Reverse codon transcript genes were designed to utilise the most frequently used codons for all amino acids in U. parvum or an exact match to the anti -codons for tRNA conserved in all U. parvum from whole genome sequence analysis. Successfully transformed U. parvum (several strains), M. agalactiae (PG2), or M. capricolum (California Kid / ATCC 27343). Previous dogma dictates that the CGG codon for arginine cannot be recognised by the available tRNAs (i.e. a dead codon), leading to stalled protein synthesis and accumulation of incomplete peptides. Therefore 1-3 codons utilising CGG or rarely used CTG codons for leucine or TCG codons for serine, were inserted and expression compared. Fluorescence and luminescence were quantified by Fluostar plate reader and in utero infected mice were visualised with CCD camera. Results: The U. parvumtufA promoter was found to be superior to the major surface protein (mba) genepromoter for gene expression and stepwise increases in fluorescence/luminescence were found with delivery of 2-4 copies of expression cassette. Fluorescence of U. parvum was too low to be useful even with 4 copies of RFP, while single copy Luc could be readily detected; however, luminescent levels (2.3 ± 0.6 x105 units) were 1000-fold lower than the same gene in agricultural mycoplasma strains (3.6 ± 1.1 x107 and 5.1 ± 0.7 x107 units). Use of CGG codon for Arginine had no significant decrease in exogenous gene expression in U. parvum, M. agalactiae or M. capricolum, while use of the CTG leucine and TCG serine codons increased expression in U. parvumand M. capricolum. Genes synthesized with matching tRNA anticodons was found to significantly decrease RFP and Luc expression only for U. parvum. Conclusions: Ureaplasma expression of exogenous genes is significantly diminished relative to other Mycoplasmas, but constructs were successfully created to enable in vivo imaging of intrauterine Ureaplasma infection in a pregnant animal model.