This study reports the detection, for the first time, of human rotavirus in stools of children and the molecular characterization of isolated circulating strains in Botswana. We collected 249 stool samples between 1999 and 2001 from children with diarrhoea in three health districts of Botswana and examined them for the presence of rotavirus antigens and particles. Group A rotavirus antigen was detected in 43 of 249 (17%) of the samples tested by enzyme-linked immunosorbent assay. Of the 43 children shedding rotaviruses, 37 (86%) were infants < or =2 years of age. The presence of rotavirus particles was also confirmed by direct electron microscopy. The characteristic 11 segments of the double-stranded RNA mobility pattern of rotavirus were demonstrated by polyacrylamide electrophoresis in 20 of 43 (47%) of the rotavirus-positive samples. The predominant electrophoretic pattern detected was the long (L) electrophoretype 14 of 20 (70%) followed by the short (S) electrophoretype five of 20 (25%). One strain had a mixed (L/S) pattern. Of the 26 samples subjected to subgrouping by enzyme immuno assay, eight were typed as subgroup-II specific and seven were subgroup I. The predominant VP7 genotypes detected were G1 (59%). Two mixed strains of G1 + G3 (5%) and G1 + G2 (5%) were also detected. VP4 genotypes in circulation were: P[4] (5%), P[6] (33%) and P[8] (33%). Mixed P-types P[4 + 6] (5%) and P[6 + 8] (18%) were also detected. Rotavirus strains G1 P[8] and GI P[6 + 8] were the most common cause of diarrhoea in our study area.