Well, the first color cannot be used in the image, as it will not be seen. It's used to mark areas where the background will show through. Due to the way that some programs change the palette, I normally use RGB value of 0,0,0 for color zero and say 4,4,4 to represent black.
I normally use 256 color PCX (with only the first 16 colors used), because that's what my tools support.
The default tile size is 16x16, so the image should be a multiple of 16x16.