Technological advancement in molecular biology techniques has proved to be useful in successful overexpression of recombinant proteins. However, one of the major issues of concern is the instability of recombinant plasmid when the recombinant hosts are grown without selection pressure. Current study proposes a correlation between segregational instability and plasmid size. In order to explain the segregational instability, existing mathematical model have been used. Four different recombinant vectors pET29b, pET32b, pET43b and pCS22, all having 1.5 kb alpha-amylase gene (amyE) insert from Bacillus amyloliquefaciens were used in the study. Culture when grown in repeated batch without suitable selection pressure showed plasmid free cells generating from plasmid bearing cells. Segregational instability of 2.2 x 10(-3) and 1.4 x 10(-3) per generation was observed with pET43b-amyE plasmid and pCS22-amyE plasmid respectively. This is the first report on segregational instability of commercially available vectors. A mathematical correlation has been proposed between plasmid size and rate of segregational loss per generation (R). (C) 2009 Elsevier B.V. All rights reserved.