Cytomegalovirus (CMV) is a significant contributor to morbidity and mortality among immunocompromised individuals; therefore, rapid and accurate diagnosis is essential. We compared positive cultures (n = 147) from different specimen types as to (a) the incubation time to a positive result and (b) the reproducibility of positive findings in replicate cultures. Five replicate shell vials were inoculated from each specimen: Two vials were stained at 24 h, two at 48 h, and one held and observed for a maximum of 30 days. Positive cultures from tissue biopsy specimens required the shortest incubation (mean = 1.9 days) and urine specimens the longest (mean = 3.9 days) (P < .005). Tissue biopsy specimens were the most reproducible (48.4% of specimens were positive in five of five replicates) and urine specimens the least (no specimens were positive in five of five replicate vials) (P < = .0002). The observed interspecimen variability is important because failure to understand and adjust for these differences could negatively influence the ability to identify CMV in culture.