We studied the dynamics of the microbial population during ripening of Cueva de la Magahá cheese using a combination of classical and molecular techniques. Samples taken during ripening of this Spanish goat's milk cheese in which Lactococcus lactis and Streptococcus thermophilus were used as starter cultures were analyzed. All bacterial isolates were clustered by using randomly amplified polymorphic DNA (RAPD) and identified by 16S rRNA gene sequencing, species-specific PCR, and multiplex PCR. Our results indicate that the majority of the 225 strains isolated and enumerated on solid media during the ripening period were nonstarter lactic acid bacteria, and Lactobacillus paracasei was the most abundant species. Other Lactobacillus species, such as Lactobacillus plantarum and Lactobacillus parabuchneri, were also detected at the beginning and end of ripening, respectively. Non-lactic-acid bacteria, mainly Kocuria and Staphylococcus strains, were also detected at the end of the ripening period. Microbial community dynamics determined by temporal temperature gradient gel electrophoresis provided a more precise estimate of the distribution of bacteria and enabled us to detect Lactobacillus curvatus and the starter bacteria S. thermophilus and L. lactis, which were not isolated. Surprisingly, the bacterium most frequently found using culture-dependent analysis, L. paracasei, was scarcely detected by this molecular approach. Finally, we studied the composition of the lactobacilli and their evolution by using length heterogeneity PCR.Lactic acid bacteria (LAB) are functionally related by their ability to produce lactic acid via either homo-or heterofermentative metabolism. The acidification and enzymatic processes accompanying the growth of LAB impart distinctive flavors and textures to a wide variety of fermented dairy products, meats, and vegetables. In addition, their preservative qualities make these bacteria useful in the control of undesirable microorganisms (16). For this reason LAB have important applications in the food, agricultural, and medical sectors and have been the subject of considerable research and commercial development over the past decade. Lactobacilli are well represented in fermented milk, yogurt, and cheese, as either natural or intentionally added microbiota (starter cultures). Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus curvatus, Lactobacillus acidophilus, and Lactobacillus pentosus are common members of the nonstarter LAB (NSLAB) communities in dairy products (11,32,35). In cheese NSLAB are represented mainly by L. casei and L. paracasei (5,11,20), but L. brevis, L. plantarum and L. curvatus are also important (4,5,43).