The production and regeneration of bacterial protoplasts promoted the loss of three different plasmid-specified traits in Streptococcus lactis subsp. diacetylactis strains. The loss of five different plasmids, including small multicopy molecules, was readily detected in Streptococcus lactis 712 by screening lysates of random protoplast regenerants on agarose gels. In this strain sequential rounds of protoplast regeneration were used to produce a plasmid-free strain and derivatives carrying only single molecules from the plasmid complement. During these experiments a 33-megadalton plasmid, pLP712, was found to encode genes for lactose and protein utilization. Only this plasmid was required for normal growth and acid production in milk; the remaining four plasmids appeared to be cryptic. Lactose-defective derivatives of a strain carrying only pLP712 were readily isolated. Although these derivatives included instances of plasmid loss, deletions of pLP712 were frequently found. Many different deleted derivatives of pLP712, including some in which the lactose or protein utilization determinant or both were lost, were isolated. The molecular instability of pLP712 largely accounted for previous observations of plasmid complements in S. lactis 712 after lactose determinant curing or transfer by conjugation and transduction. Curing of cryptic molecules from multiple plasmid complements by protoplast regeneration may prove to be generally valuable in lactic streptococci and other gram-positive species.