Temporal variation in allozyme frequencies at six loci was studied by making monthly collections over 4 yr in one population of the cactophilic species Drosophila buzzatii. Ten sites were defined within the study locality, and for all temporal samples, separate collections were made at each of these sites. Population structure over microgeographic space and changes in population structure over time were analyzed using F-statistic estimators, and multivariate analyses of allele and genotype frequencies with environmental variables were carried out. Allele frequencies showed significant variation over time, although there were no clear cyclical or seasonal patterns. A biplot analysis of allele frequencies over seasons within years and over years showed clear discrimination among years by alleles at four loci. During the 4 yr, three alleles showed directional changes which were associated with directional changes in environmental variables. Significant associations with one or more environmental variables were found for allele frequencies at every locus and for both expected and observed heterozygosities (except those for Est-1 and Est-2). Thus, variation in allele frequencies over time cannot be attributed solely to drift. Significant linkage disequilibria were detected among three loci (Est-2, Hex and Aldox), but there was no evidence for spatial or temporal patterns. The F-statistic analyses showed significant differentiation among months within years for all loci, but the statistic used (coancestry) was heterogeneous among loci. Estimates of F (inbreeding) for all loci were significantly different from zero, with the loci in four groups, Adh-1 (negative), Pgm(small positive), Est-2 and Hex (intermediate) and Est-1 and Aldox (high positive). The correlation of genes within individuals within populations (f) for each locus in each month by site sample differed among loci, as did the (f) for each locus in each month by site sample differed among loci, as did the patterns of change in f over time (seasons). Heterogeneity in the F-statistic estimates indicates that natural selection is directly or indirectly affecting allele and genotype frequencies at some loci. However, the F-statistic analyses showed essentially no microgeographic structure (i.e., among sites), although there was significant heterogeneity in allele frequencies among flies emerging from individual rots. Thus, microspatial heterogeneity probably is most important at the level of individual rots, and coupled with habitat selection, it could be a major factor promoting diversifying selection and the maintenance of polymorphism.(ABSTRACT TRUNCATED AT 400 WORDS)