The optimal culture conditions for Bacillus stearothermophilus NUB3621 (BGSC 9A5) in chemostat were studied. The results obtained showed that the optimal culture conditions in terms of biomass concentration and maximum growth rate were 65 degrees C, pH 6.8 to 7.2. Dissolved oxygen became growth limiting at pO(2) levels below 10%. Furthermore, this strain was transformed with three new hybrid vectors (pPAM2, pPCH2, or pPLY2) constructed by cloning in pRP9, a plasmid based on the thermophilic replicon, pBC1, and three heterologous genes : the alpha-amylase gene from Bacillus licheniformis, the cholesterol oxidase gene from Streptomyces sp., and the lipase gene from Pseudomonas fluorescens. The influence of several fermentative conditions on segregational and structural stability of the recombinant B. stearothermophilus NUB3621 transformants was studied. The parameters of plasmid loss, that is, rate of plasmid loss (R) and specific growth rate difference (delta mu), were calculated. B. stearothermophilus NUB3621 carrying pRP9 showed great segregational stability in all the assayed conditions, exceeding more than 300 generations without significant plasmid loss, whereas NUB3621 carrying pPAM2, pPCH2, or pPLY2 exhibited relatively low plasmid stability. The segregational instability of the recombinant constructs increased by increasing the fermentation temperature, decreased by increasing the dilution rate, and was not affected by the level of dissolved oxygen. On the other hand, plasmid maintenance decreased in minimal medium if compared with the results obtained in complex medium. Restriction analyses carried out on cultures of NUB3621 carrying pRP9, pPAM2, pPCH2, or pPLY2, grown for 200 generations on nonselective media, revealed that all the clones tested contained the parental plasmids. These results indicate that the heterologous inserts did not affect the structural stability of the recombinant plasmids.