In Spiroplasma citri gene inactivation through homologous recombination has been achieved by using the replicative, oriC plasmid pBOT1 as the disruption vector. However, plasmid recombination required extensive passaging of the transformants and, in most cases, recombination occurred at oriC rather than at the target gene. In the current study, we describe a new vector, in which the oriC fragment was reduced to the minimal sequences able to promote plasmid replication. Using this vector to inactivate the motility gene scm1 showed that size reduction of the oriC fragment did increase the frequency of recombination at the target gene. Furthermore, to avoid extensive passaging of the transformants, we developed a strategy in which the selective, tetracycline resistance phenotype can only be expressed once the plasmid has integrated into the chromosome by one single crossover recombination at the target gene. As an example, targeting of the spiralin gene is described.