Endogenous chromosomal DNA amplifications with associated tetracycline resistance (Tc(r)) in Bacillus subtilis were first described by C.R. Wilson and A.E. Morgan (J. Bacteriol. 163:445-453, 1985). We have confirmed and extended these results, and we show that fusion of protoplasts from Tc(s) B. subtilis 168 trpC2 with polyethylene glycol and regeneration on medium containing 20 μg of tetracycline per ml induces Tc(r) regenerants that contain amplified DNA. This phenomenon appeared to be recE dependent and requires the addition of polyethylene glycol. Along with three regenerants kindly provided by Wilson and Morgan (RAD1, RAD6, and RAD7), we characterized three strains (CLI20, CLI22, CLI30) isolated in this laboratory. All six contain an amplified region of DNA which was independently cloned on plasmid pCIS7. Integration of pCIS7 into the wild-type (Tc(s)) B. subtilis chromosome and amplification of the plasmid sequences generated a Tc(r) phenotype, even though the DNA on pCIS7 was cloned from Tc(s) B. subtilis KS162 (Ives and Bott, J. Bacteriol. 171:1801-1810, 1989). The amplified DNA also showed homology (through hybridization analysis) with pAMα1Δ1, a gram-positive Tc(r) plasmid, indicating that B. subtilis normally contains a silent integrated copy of the gene whose amplification confers Tc(r). The amplifications were determined to lie between purA and gyrB on the B. subtilis chromosome, and the endpoints were mapped. RAD6 and CLI30 may share the same left-hand endpoint, but the other endpoints are different in each isolate. The amplified DNAs of RAD1, RAD6, CLI20, and CLI30 end near known DNA membrane binding sites. The number of amplified units of DNA was determined through dot blot analysis to be approximately 80 to 100 copies per cell, with corresponding increases in transcription of RAD1, RAD6, CLI20, CLI22, and CLI30.