Despite the widespread application of the random amplified polymorphic DNA (RAPD) technique, there is no experimental evidence of the molecular mechanism of random amplification starting from a complex template. To investigate this mechanism, we cloned and sequenced 23 selected RAPD bands amplified from Haemophilus influenzae Rd genomic DNA using eight decamer primers different in GC content and/or nucleotide sequence. As the whole genome sequence of H. influenzae Rd has been reported, the exact nucleotide sequence of each primer-template annealing site was identified. Results showed that, on an average, a homology of eight base pairs was involved in priming events and that the number of nonhomologous base pairings declined exponentially from the 5' end of the primer to its 3' end. The interaction between the primer and the template DNA was stabilized by the formation of secondary structures, and a perfect match of the 3' terminal region of the primer was not necessary for successful amplification. The complexity of the annealing process suggested that, in the studied reaction conditions, many primer-template annealing sites were extended in the first cycles and that differences in the efficiency of priming and replication processes led to amplification of RAPD fragments. Moreover, the distribution of the amplified regions on the H. influenzae chromosome was analyzed.