Single-molecule fluorescence spectroscopy was used to probe the lateral transport of an adsorbed oligonucleotide, the SP6 promoter primer, which is a 24-mer labeled at the 5' end with tetramethylrhodamine. The oligonucleotide was adsorbed to the interface of an aqueous solution of 0.01 M KC1 and silica chemically modified by chlorodimethyloctadecylsilane. Confocal fluorescence microscopy achieved single-molecule resolution, with a molecule in the beam 7% of the time. Autocorrelation of the data fit well to a model having two species, one diffusing and the other one undergoing transient strong adsorption. A small number of bursts, 0.3%, had unusually long durations, accounting for the slow component in the autocorrelation. When these long bursts were excised from the data, the autocorrelation fit well to simple diffusion, with D = 4 x 10(-6) cm(2)/s. Autocorrelation of the long bursts alone gave a rate constant for desorption of 3 s(-1). The strongly adsorbed molecules were found to comprise 10% of the total population of adsorbates. It is concluded that the lateral transport of the SP6 promoter primer is described by fast lateral diffusion interrupted by rare, reversible, strong adsorption to defect sites. During strong adsorption, the tetramethylrhodamine label undergoes hindered motion, suggesting it is not the adsorbing moiety.