We describe a study designed to assess properties of a P300 brain-computer interface (BCI). The BCI presents the user with a matrix containing letters and numbers. The user attends to a character to be communicated and the rows and columns of the matrix briefly intensify. Each time the attended character is intensified it serves as a rare event in an oddball sequence and it elicits a P300 response. The BCI works by detecting which character elicited a P300 response. We manipulated the size of the character matrix (either 3 x 3 or 6 x 6) and the duration of the inter stimulus interval (ISI) between intensifications (either 175 or 350 ms). Online accuracy was highest for the 3 x 3 matrix 175-ms ISI condition, while bit rate was highest for the 6 x 6 matrix 175-ms ISI condition. Average accuracy in the best condition for each subject was 88%. P300 amplitude was significantly greater for the attended stimulus and for the 6 x 6 matrix. This work demonstrates that matrix size and ISI are important variables to consider when optimizing a BCI system for individual users and that a P300-BCI can be used for effective communication.