The aim of this study was to develop a high-speed digital imaging system and related software for ciliary beat frequency (CFB) analysis in order to establish an automated and reliable method that is observer independent and faster compared to the conventional computerized microscope photometry method. Using primary human nasal epithelial cell cultures, the CBF was recorded with a computerized microscope photometry system and a high-speed digital imaging system. To obtain a wide range of frequencies, glycocholate (0.5%) and chlorocresol (0.005%) were used as ciliostimulatory and cilio-inhibitory reference compounds, respectively. The mean values in hertz (+/- s.d.) obtained with the photometry and high-speed digital imaging systems were: controls 8.2 +/- 0.9 and 7.9 +/- 1.1; chlorocresol 5.0 +/- 0.9 and 5.1 +/- 1.1; glycocholate 9.8 +/- 1.0 and 9.7 +/- 0.8. A similar increase (by 20 and 24%) and decrease (by 38 and 35%) in CBF was determined by the two methods after glycocholate and chlorocresol treatment, respectively. The mean difference between the photometry and high-speed digital imaging methods was 0.2 +/- 0.6 Hz, and the Bland-Altman limits of agreement were from -1.0 to +1.4 Hz, suggesting that the results obtained by these two methods could be used interchangeably. These results show the reliability of the high-speed digital imaging system and the software developed for in-vitro CBF measurements. The advantages of the system include: (i) fast data acquisition and calculation, (ii) whole field automated CBF analysis and (iii) reduction in selection bias.