Malaria transmission was assessed in two rural communities, Kona and Afamanaso in Sekyere South district, Ashanti Region, in the forest zone of Ghana to provide baseline data for ongoing clinical studies and the evaluation of the effect of interventions. Altogether, 3,479 Anopheles gambiae and 1,157 Anopheles funestus were caught by human landing catches. Sporozoite rates determined by either microscopy of salivary glands or enzyme-linked immunosorbent assay (ELISA) for Plasmodium falciparum in the two villages were 6.6% vs. 8.9% for the main vector A. gambiae and 3.2% vs. 6.3% for A. funestus. ELISA tests of dissected specimens compared to microscopy of salivary glands were 1.3 and 2.0 times more positive for A. gambiae and A. funestus, respectively. Plasmodium infections of 122 microscopically positive salivary glands of A. gambiae were identified by real-time PCR as 95 (77.9%) P. falciparum, 7 (5.7%) Plasmodium malariae, 7 (5.7%) Plasmodium ovale and 1 (0.8%) mixed infection of P. falciparum and P. malariae. Transmission in the area was found to be intense and perennial with some seasonal variations during the study period from Dec. 2003 to Aug. 2005. Although the two villages were only 10 km apart from each other, Annual Biting Rates (ABRs) and Annual Entomological Inoculation Rates (AEIRs) were much higher at Afamanaso (11,643 vs. 866) than at Kona (5,329 vs. 490). Most of the transmission (91.4%) occurred during bedtime hours from 21 to 6 h. It is important to note that there was still a substantial transmission before 21 h with AEIRs of 57.3 at Afamanso and 38.7 at Kona. The distribution of impregnated bednets alone, therefore, may not be sufficiently effective.